Pregnancy diagnosis in the domestic horse through direct urinary estrone conjugate analysis

Urinary estrone conjugates were measured directly by radioimmunoassay (RIA) in 20 pregnancies from preconception diestrus to day 78 of pregnancy. High performance liquid chromatography separation defined estrone sulfate (ES) as the predominant immunoreactive peak which accounted for 94% to 97% of the total immunoreactivity after chromatography. Diestrous values indexed by creatinine were 0.15 ± 0.07 micrograms/mg Cr, $\text{x}\text{± SEM}$ as compared to estrous values which rose to 0.47 ± 0.14 micrograms/mg Cr, $\text{x}\text{± SEM}$. Urinary ES concentrations significantly increased (P = 0.0001 in pregnant mares from day 35 to day 47 (1.21 ± 0.12 micrograms/mg Cr) as compared to day 25 to day 34 (0.27 ± 0.01 micrograms/mg Cr). Measurement of urinary ES may provide an alternate or augmentive method of pregnancy diagnosis in the domestic mare.     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

Serum and urinary estrone sulfate during the menstrual cycle, measured by a direct radioimmunoassay, and fate of exogenously injected estrone sulfate

Serum and early-morning urinary levels of estrone sulfate during the menstrual cycle were measured by a direct radioimmunoassay without hydrolysis. These levels were high and showed prominent peaks [serum, 2.67 +/- 0.37 ng/ml (mean +/- SE); urine, 5.82 +/- 2.3 micrograms/l] around the day of the preovulatory estradiol-17 beta peak, and increased again during the luteal phase. Following intravenous injection of estrone sulfate, serum estrone sulfate, estrone and estradiol-17 beta were measured. The conversion of estrone sulfate to estrone and/or estradiol-17 beta was very small during their transit in the general circulation.     

Urinary Progestogen and Estrogen Excretion During Pregnancy in Eulemur macaco flavifrons,E. rubriventer,and Hapalemur griseus occidentalis

Via a non-invasive approach, we aimed to provide comparative data on the presence and relative abundance of progestogen and estrogen metabolites excreted into the urine of Eulemur rubriventer, E. macaco flavifrons and Hapalemur griseus occidentalis and to characterize the patterns of progestogen and estrogen excretion during pregnancy. We found that estrone is the major urinary estrogen in Eulemur macaco flavifrons and Hapalemur griseus occidentalis, while 16-hydroxyestrone appeared to be the predominant estrogen in the urine of E. rubriventer. Estradiol-17ß was either absent (Hapalemur) or present in very low amounts. HPLC of progestogens had high levels of immunoreactivity in an assay against 5-pregnane-3-ol-20-one (5-P-3OH) in each species, though the nature of the progestogens measured by the 5-P-3OH assay differed among species. During pregnancy, 5-P-3OH levels remained relatively low in Eulemur rubriventer, whereas in E. macaco flavifrons and Hapalemur griseus occidentalis, a sustained elevation in levels occurred from mid-pregnancy until the final weeks before parturition. Estrogen excretion differed depending on the sex of the fetus in each species, with only females carrying male infants showing clearly elevated estrogen levels during the last 6–8 weeks of gestation. In conclusion, we demonstrate both species similarities and differences in the metabolism and urinary excretion patterns of reproductive hormones throughout gestation in the Lemuridae. The data not only extend our knowledge on the reproductive physiology of lemurs but also show that more studies on other lemur taxa are needed to provide a broader basis for interspecific comparison.     

The development and application of an enzyme immunoassay for urinary estrone conjugates

An enzyme immunoassay (EIA) for estrone conjugates is described and applied to urine samples from a female Indian rhinoceros, a female gorilla, and a female lion-tailed macaque. Concomitant measures of estrone conjugates in the same sample are compared to the values obtained with radioimmunoassay. High correlation coefficients for values obtained from each assay indicate that EIA measurements provide information that is comparable to values obtained by radioimmunoassay. EIA methods for urinary steroid conjugates can provide a practical tool to evaluate female reproductive status of zoo species without the need for a traditional endocrine laboratory.     

The relationship between female urinary estrogen excretion and mating behavior in cotton-topped tamarins,Saguinus oedipus oedipus

Daily urinary estrogen excretion in six singly housed adult female cottontopped tamarins was measured by radioimmunoassay. Each female was paired with a male for 1 hr every day, during which behavioral observations were conducted on the pair from behind a oneway glass screen. During each observation period the duration or frequency of occurrence of various behaviors was recorded. In this paper, the daily variations of (1) total frequency of male mounting, (2) total frequency of male mounting with thrusting, and (3) latency to first male mount are presented in relation to the measured pattern of female estrogen excretion. The results reveal a high degree of behavioral variation between pairs, although a relationship between rhythmic changes in sexual activity and female estrogen excretion in individual pairs is apparent. Mating occurs throughout the female cycle and also during at least the first 2–3 weeks of pregnancy. A gestation period of 170–172 days is estimated in respect of one female, in agreement with a previous observation of one gestation of at least 166 days. The high frequency and intensity of mating recorded in this controlledintroduction study give support to the proposition that mating is important in pairbonding in this monogamous species. It is concluded that under the experimental procedure followed, mating behavior did not reliably reflect the female’s reproductive hormonal state.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: V. Estrogen and pregnanediol-3-glucuronide excretion in the black rhinoceros (Diceros bicornis)

A study of female black rhinoceros (Diceros bicomis) urinary steroid and steroid metabolite excretion was performed to determine if techniques useful for monitoring reproductive events in the Indian rhinoceros (Rhinoceros unicornis) could be utilized to evaluate the black rhinoceros. Urine samples from 19 zoo-held black rhinoceros were analyzed for estrogen, estrone conjugates (EC), and pregnanediol-3-glucuronide (PDG) content by direct radioimmunoassays. Estrogen analysis revealed that >95% of the estrogens present in female black rhinoceros urine are conjugated, with estrone and estradiol accounting for virtually all of these estrogens. There is no observable difference in the amount of estrogen present in estrus; postestrus; and early-, mid-, and late-gestation urine samples. Analysis of serial urine samples for EC failed to reveal any discernible levels or patterns which reflected reproductive status. Neither nonpregnant nor early-gestational female black rhinoceros' urine samples contained detectable amounts of PDG. Urinary PDG concentrations became measurable in midgestation (9–12 months prior to parturition) and rose steadily throughout the remainder of gestation. PDG levels declined sharply and became nondetectable 1 day postpartum. Though a wide range in PDG levels was observed among individual pregnant animals, each female consistently excreted increasing amounts of PDG through latter pregnancy.     

Assessing reproductive status in orang-utans by using urinary estrone

The aim of this work was to compare normal menstrual cycle patterns of urinary estrone excretion in orang-utans with those of an orang-utan in her late 40s, an orang-utan before and after rehabilitation for anorexia, and an orang-utan during prolonged lactation. A detailed menstrual and sexual activity record was kept for each orang-utan subject. Urine samples for estrone radioimmunoassay were collected daily for two cycles whenever possible. “Mawas,” in her late 40s, was monitored for two cycles in each of 3 successive years. While the normal pattern of estrone excretion was initially seen, some of the later cycles were shorter and appeared to lack the large luteal-phase peak. “Puspa,” anorexic and amenorrhoeic at the age of 9½ years, had very low urinary estrone concentrations. After rehabilitation, followed by a late menarche, she had regular menstrual cycles with a normal estrone excretion pattern. She gave birth to a normal infant in 1990. “Puteri,” who had been lactating for 2 years, had consistently low estrone concentrations during an 8-week monitoring period, with no evidence of cyclic variation in estrone excretion. Daily urinary estrone measurements, together with observation, were found to be useful in assessing the reproductive status of orangutans.     

Direct measurements of urinary estrone conjugates during the normal menstrual cycle of the gorilla (Gorilla gorilla)

A direct immunoassay for urinary estrone conjugates (estrone sulfate and estrone glucuronide) was used to assess the preovulatory estrogen rise in normal gorilla menstrual cycles. Immunoreactive estrone conjugates in samples concomitantly assessed for total estrogen immunoreactivity reflected similar profiles throughout the cycle; however, the speed and resolution of the direct assay for conjugates indicate this method to be more accurate in monitoring ovulation than the measurement of total immunoreactive estrogens. In a single conceptive ovarian cycle, urinary estrone conjugate continued to rise in the luteal phase, indicating that this test may also be useful for detecting early pregnancy. The application of this technique provides clear profile of ovarian function in gorillas as well as in other primate species.     

Comparative analysis of urinary biomarkers for early detection of acute kidney injury following cardiopulmonary bypass

The purpose of this study was to compare the performance of six candidate urinary biomarkers, kidney injury molecule (KIM)-1, N-acetyl-β-D-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), interleukin (IL)-18, cystatin C and α-1 microglobulin, measured 2?h following cardiopulmonary bypass (CPB) for the early detection of acute kidney injury (AKI) in a prospective cohort of patients undergoing cardiac surgery. A total of 103 subjects were enrolled; AKI developed in 13%. Urinary KIM-1 achieved the highest area under-the-receiver-operator-characteristic curve (AUC 0.78, 95% confidence interval 0.64–0.91), followed by IL-18 and NAG. Only urinary KIM-1 remained independently associated with AKI after adjustment for a preoperative AKI prediction score (Cleveland Clinic Foundation score; p?=?0.02), or CPB perfusion time (p?=?0.006). In this small pilot cohort, KIM-1 performed best as an early biomarker for AKI. Larger studies are needed to explore further the role of biomarkers for early detection of AKI following cardiac surgery.     

Changes in plasma levels of estrone sulfate and estrone in the pregnant ewe around parturition

A method for the extraction, separation and measurement of estrone sulfate and estrone in a single plasma sample is described. The method has been applied to the determination of plasma levels of estrone sulfate and estrone in pregnant ewes over the period 60 hr before to 20 hr after parturition. The study revealed that the plasma levels of estrone sulfate and estrone began to increase about 40 hr before parturition, reached a peak at parturition and then declined rapidly to levels below the sensitivity of the method by 15 hr postpartum. The peak level of estrone sulfate recorded at parturition was 103 pmol (38 ng) per ml of plasma which was approximately 30 times greater than the corresponding peak level of estrone.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: III. Estrone sulfate and pregnanediol-3-glucuronide excretion in the Indian rhinoceros (Rhinoceros unicornis)

A study was undertaken to identify urinary estrogen and progesterone metabolites in the female Indian rhinoceros (Rhinoceros unicornis). Measurements of these metabolites were then used to monitor ovarian function and establish normal levels and patterns of steroid excretion during the estrous cycle and pregnancy. Urine samples were analyzed for estrone sulfate and pregnanediol-3-glucuronide (PDG) by direct radioimmunoassays. Both hormones produced discrete profiles reflecting ovarian activity in nonconceptive cycles. The estrous cycle was observed to be 48 days (range 39–64) with a mean follicular phase of 14.8 days (range 13–19), followed by a mean luteal phase of 19 days (range 17–21). Of the single gestation monitored, PDG levels rose above luteal phase levels by the third month after breeding and remained elevated throughout gestation. The combined estrogen and progesterone metabolite profiles present a complete evaluation of ovarian steriod production in the mature female Indian rhinoceros.     

The detection of ovulation and early pregnancy in the baboon by direct measurement of conjugated steroids in urine

The pattern of excretion of urinary steroid metabolites in the olive baboon (Papio anubis) was examined during the menstrual cycle and in conception cycles in which embryos were surgically removed at intervals between day 11 and day 21 (day 0 = day of preovulatory estrogen peak). Conjugated estrone and pregnanediol-3α-glucuronide were measured in overnight urine samples by direct, nonextraction assays, and the levels were indexed by creatinine. Results showed that measurement of urinary conjugated estrone reflected preovulatory estrogen output and that pregnanediol-3α-glucuronide was an abundant urinary metabolite of progesterone. There was a defined postovulatory increase in the excretion of conjugated estrone during conception cycles in eight of ten animals. The timing of the increase ranged between day 13 and day 19 and was related to the appearance of elevated levels of urinary gonadotrophin. In four animals, increased estrogen excretion was first detected after the day of embryo removal, but this was most likely a response to chorionic gonadotrophin secreted before surgery. The findings demonstrate that measurement of conjugated estrone offers a rapid and practical approach for monitoring ovulation and implantation in the baboon by a single assay technique.     

Atypical cells in the urinary sediment: a protocol for cytological analysis of the urinary sediment

Introduction:  The Department of Clinical Chemistry of the General Hospital of Móstoles (Madrid, Spain) has for many years diagnosed the presence of atypical cells in the urinary sediment (ACUS), but this finding only led to a comment in the final report of the urinalysis, informing the clinician of this fact in case he/she decided to perform further studies in these patients.
Methods:  Since 2004, the Clinical Chemistry Department has implemented a new protocol together with the Department of Surgical Pathology of the Hospital, according to which all the urinary sediment samples with ACUS are sent for cytological analysis. In this report, we comment on the results corresponding to 99 samples.
Results:  With this new strategy, we can directly diagnose if the ACUS correspond to a carcinoma or to another process, and this has allowed us to reduce time until diagnosis in our patients and also to avoid unnecessary studies in negative cases. We comment on our results with this new management strategy.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: VI. Pregnancy detection in exotic equidae

The measurement of serum and urinary estrone sulfate (ES) was evaluated on random single samples for the detection of pregnancy in the Przewalski's horse (PRZ) and Hartmann's mountain zebra (HMZ). Pregnancy was detected by ES analysis of urine or serum in the PRZ and HMZ by at least 8 and 10 months before delivery, respectively. The values of ES in both serum and urine are shown at various stages of gestation. Results indicate urinary ES analysis is a noninvasive early diagnosis for pregnancy in these species.     

Studies of sodium channels in rabbit urinary bladder by noise analysis

Summary Sodium channels in rabbit urinary bladder were studied by noise analysis. There are two components of short-circuit current (I _sc) and correspondingly two components of apical Na⁺ entry, one amiloride-sensitive (termedI _A and the A channel, respectively) and one amiloride-insensitive (I _L and the leak pathway, respectively). The leak pathway gives rise tol/f noise, while the A channel in the presence of amiloride gives rise to Lorentzian noise. A two-state model of the A channel accounts well for how the corner frequency and plateau value of Lorentzian noise vary with amiloride concentration. The single-channel current is 0.64 pA, and the conducting channel density is on the order of 40 copies per cell. Triamterene blocks the A channel alone, and increasing external Na⁺ decreases the number but not the single-channel permeability of the A channel. Hydrostatic pressure pulses (punching) increase the number of both pathways. Repeated washing of the mucosal surface removes most of the leak pathway without affecting the A channel.Properties of the A channel revealed by noise analysis of various tight epithelia are compared, and the mechanism ofl/f noise is discussed. It is suggested that the A channel is synthesized intracellularly, stored in intracellular vesicles, transferred with or from vesicular membrane into apical membrane under the action of microfilaments, and degraded into the leak pathway, which is washed out into urine or destroyed. The A channel starts withP _Na/P _K30 and loses selectivity in stages untilP _Na/P _K reaches the free-solution mobility ratio (0.7) for the leak pathway. This turnover cycle functions as a mechanism of repair and regulation for Na⁺ channels, analogous to the repair and regulation of most intracellular proteins by turnover. Vesicular delivery of membrane channels may be operating in several other epithelia.     

Monitoring ovarian function in marmosets and tamarins by the measurement of urinary estrogen metabolites

Practical aspects of urinary estrogen analysis were considered with regard to establishing simple and reliable methods for monitoring ovarian function in marmosets and tamarins. Changes in the hormone:creatinine ratio in small volumes of urine from the common marmoset were significantly correlated with changes in 24-h excretion. Comparison of the metabolism and excretion of estrogens during the ovarian cycle in the common marmoset and cottontop tamarin revealed interesting species differences. High concentrations of conjugated estrone were measured in marmoset plasma, but estradiol 17β was the predominant estrogen in urine. In contrast, estrone was the most abundant estrogen measured in tamarin urine. Both species excreted very little estriol. Sulfates and glucuronides were present in urine in similar proportions before ovulation in the marmoset, although after ovulation sulfates were the more abundant. Conversely, most of the estrogens in tamarin urine appeared to be conjugated as glucuronides. Direct assay for estrone sulfate was applied to the measurement of urinary estrogen excretion during the ovarian cycle in a marmoset. The results compared well with those for total estradiol 17β after hydrolysis and ether extraction. The use of direct assays for conjugated estrogens in small volumes of urine is suggested as a practical method for monitoring ovarian function in marmosets and tamarins.     

重组人糖基化尿型纤溶酶原激活剂中试产品抗原特异性的分析

尿型纤溶酶原激活剂（ｕ－ＰＡ）是新一代的基因工程溶栓药物。建立了重组人糖基化尿型纤溶酶原激活剂的中试生产工艺。中试产品的结构正确性和一致性分析是证明产品质量的重要依据,所以,对三批产品进行了抗原特异性分析。结果,ｕ－ＰＡ三批中试产品ＳＤＳ－ＰＡＧＥ显示的蛋白区带与免疫印迹显示的免疫活性区带没有批次判别说明ｕ－ＰＡ三批中试产品表现了一致的ｕ－ＰＡ的免疫原性。     

Pregnancy diagnosis in sows: direct ELISA for estrone in feces and its prospects for an on-farm test, in comparison to ultrasonography

The usefulness of fecal estrone (E1) measurement as a tool for pregnancy diagnosis was investigated. Concentrations of E1 were measured in feces from pregnant and nonpregnant sows by a direct ELISA without extraction. Highly significant differences in E1 concentrations were found in feces from nonpregnant and pregnant sows (P = 0.016 to < 0.001). Pregnancy diagnosis on Days 26 to 32 after insemination, based both on fecal E1 concentrations as measured by ELISA and ultrasonography using a 5.0 MHz linear-array transducer, was performed in a group of 496 gilts and sows. The fecal E1 test had a sensitivity (correct diagnosis of pregnancy) of 96.5% and a specificity (correct diagnosis of nonpregnancy) of 93.6%, using 3.65 ng E1/g feces as a cut-off value. For ultrasonographic pregnancy diagnosis the test sensitivity and specificity were 99.3 and 92.5%, respectively. Although an increase of fecal E1 concentrations was noticed for increasing litter sizes, the results indicated that these concentrations could not be used to predict litter size. It is concluded that the distribution of fecal E1 concentrations in both nonpregnant and pregnant sows offers a suitable basis for the development of a simple, sow-side pregnancy test.     

Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC₅₀ below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC₅₀ below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 μM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.