Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

The radioimmunoassay of steroid glucuronides. The oestrogen C-3 glucuronides as haptens.

Antisera were prepared against three related oestrogen ring-A glucuronides, oestrone 3-glucuronide, oestradiol 3-glucuronide and oestriol 3-glucuronide. The corresponding 6,7-3H-labelled conjugates were synthesized as radioligands and the cross-reactions of the antisera against ring-A oestrogen glucuronides and other steroid conjugates were examined. The specificity of the antiserum against oestriol 3-glucuronide was compared with that raised against oestriol 16alpha-glucuronide, and the measurement of the former conjugate in late-pregnancy urine is discussed.     

Use of monoclonal antibodies to pregnanediol-3α-glucuronide for the development of a solid phase chemiluminescence immunoassay

Monoclonal antibodies to pregnanediol-3α-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specifity and affinity to pregnanediol-3α-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3α-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3α-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r=0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.     

Measurement of glucuronometabolites of 17 beta-estradiol and progesterone in diluted overnight urine. An approach to the study of luteal insufficiency

The determination of the concentrations of estrone-3-glucuronide, pregnanediol-3-glucuronide and luteinizing hormone has been performed in early morning urine samples of 14 normal menstruating women using a timed and measured volume urine collection procedure. In order to investigate the variability of the urinary hormonal concentrations due to day-to-day differences in diuresis, the absolute hormonal concentrations have been corrected either for the urinary creatinine excretion or for the volume of urine voided during the night. The results demonstrate that both correction factors are able to reduce substantially the coefficient of variation values in comparison to the absolute hormonal concentrations. The urinary test of ovarian function has been performed in 11 infertile women affected by luteal insufficiency using the same procedure, and the hormonal profiles showed some alterations in both estrone-3-glucuronide and pregnanediol-3-glucuronide concentrations in comparison to the hormonal profiles of the normal subjects. Such alterations were significant in the single subject when integrated values of the hormonal data in defined time intervals were investigated.     

Development of a direct microplate enzyme immunoassay for the determination of pregnanediol-3 alpha-glucuronide in urine

A sensitive direct enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was developed. The assay system involves the use of an antiserum against pregnanediol-3 alpha-glucuronide and an enzyme-labelled antigen chemically prepared by linking beta-D-galactosidase to 20 alpha-hydroxy-5 beta-pregnane 3(O-carboxymethyl)oxime. Free from antibody-bound antigen was separated by a solid-phase double antibody method, using a microplate coupled with goat anti-rabbit gamma-globulin. This solid-phase enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was validated in terms of specificity, accuracy and sensitivity. When urine samples were assayed for pregnanediol-3 alpha-glucuronide, the results obtained by the solid phase enzyme immunoassay and conventional radioimmunoassay methods agreed well (n = 30, r = 0.922). This assay system has an advantage over radioimmunoassay, because it does not require the use of radioisotopes. The procedure of this method is very simple, since it does not require purification steps of the biological samples.     

The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites

We describe the principles of a new generation of sequential or simultaneous time-resolved fluoroimmunoassays, namely, simple, rapid, liquid-phase non-separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone-3-glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone-3-glucuronide. The antibody-binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-BSA and an aliquot of standard or sample (undiluted urine; 10 μl) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody-free label is measured in a time-resolved fluorometer and is proportional to the concentration of estrone-3-glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.     

Monoclonal antibodies to pregnanediol-3-glucuronide: application to a direct enzyme-linked immunosorbent assay of urine

Monoclonal antibodies to pregnanediol-3-glucuronide were produced and characterized. One of three clones investigated provided antibody suitable for a direct urinary enzyme-linked immunosorbent assay (ELISA). The ELISA uses a pregnanediol-thyroglobulin conjugate adsorbed onto the wells of a standard 96-well microtiter plate. Pregnanediol-3-glucuronide in standards or diluted urine competes with the immobilized steroid for antibody-binding sites. After washing, mouse monoclonal antibody bound to the plate is probed with antimouse immunoglobulin peroxidase. After further washing, o-phenylenediamine substrate is added and, finally, the absorbance is read at 492 nm. The ELISA shows excellent performance and agreement with the previous gas chromatographic method. The ELISA is ideal for aiding the assessment of ovarian function in the routine laboratory.     

The isolation of estriol-16α-glucuronide and pregnanediol-3α-glucuronide from late pregnancy urine

A simple procedure is described, whereby estriol-16α-glucuronide and pregnanediol-3α-glucuronide have been isolated from late pregnancy urine. The conjugates isolated were used as haptens for raising antisera for radioimmunoassay.     

Multiple immunoassay: The simultaneous measurement of two urinary steroid glucuronides as an index of ovarian function

Ovarian function in women may be monitored effectively by a simple, solid-phase, multiple immunoassay for the simultaneous measurement of estrone-3-glucuronide and pregnanediol-3α-glucuronide in diluted urine. IgG fractions of the respective antisera are passively adsorbed to the walls of polypropylene tubes. The labelled antigens are estrone-3-glucuronyl-6-aminoethyl-ethyl-isoluminol and [6,9-³H]-pregnanediol-α-glucuronide. Daily samples of early morning urine are diluted in buffer (1:200; v/v), and 200μl removed, in duplicate, for assay. After the binding reaction (18 h at 4°C), the solution is removed by aspiration. The antibody-bound fraction is washed twice with buffer (300 μl) containing 0.05% Tween 20. Sodium hydroxide (2 N, 300 μl) is added and the mixture incubated for 60 min at 22°C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10s. Subsequently, liquid scintillation fluid (4ml) is added to the tube and the radioactivity measured. The unknown values are determined from appropriate calibration curves. The combined method has similar sensitivitiy, accuracy, precision and clinical utility to the values obtained from the separate measurement of the two analytes.     

Measurement of estrone-3-glucuronide and pregnanediol-3α-glucuronide in early morning urine samples to monitor ovarian function

The determination of the concentration of estrone-3-glucuronide and pregnanediol-3α-glucuronide has been performed by a chemiluminescent immunoassay in early morning urine samples of 14 normal menstruating women and 11 women affected by luteal phase defect. The early morning urine samples were daily collected for an entire menstrual cycle. We have employed a timed and measured volume collection procedure as correction factor. The integrated values of the hormonal data in definite time intervals were used to create a nomogram. By means of this method, it was possible to completely separate normal from luteal insufficiency subjects and to distinguish two different types of luteal phase defects. Moreover, the same approach was applied to the study of the role and the frequency of luteal phase defect in 15 patients affected by habitual abortion and in 17 premenopausal women who had undergone quadrantectomy for T1a No Mo breast cancer. A luteal phase defect was detected in nine of the aborting patients (60%) and in eight women affected by breast cancer (47%). Finally estrone-3-glucuronide was measured in early morning urine samples of 96 prepubertal and pubertal girls in different pubertal stages and in one patient affected by precocious puberty, before and during an agonist GnRH treatment. The urinary test of ovarian function seems to be suitable for diagnostic purposes and for clinical studies.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: VII. Urinary progesterone metabolites in the Equidae assessed by immunoassay

A direct enzyme immunoassay (EIA) for non-specific urinary progesterone (Po) metabolites, utilizing a non-specific monoclonal antibody against pregnanediol-3-glucuronide, was evaluated for the purpose of assessing luteal function in equids. Urinary pregnanediol-3-glucuronide (PdG) and immunoreactive PdG-like conjugate (iPdG) concentrations, indexed by creatinine, were compared to plasma Po concentrations in non-conceptive ovarian cycles through two ovulations in four mares. High-performance liquid chromatography (HPLC) of urine from lutealphase mares and a pregnant zebra revealed an absence of significant concentrations of PdG and the presence of at least three immunoreactive compounds, all of which were more polar than PdG. The concentration of iPdG in the mare ranged from a nadir of approximately 3 ng/mg Cr at the time of ovulation to nearly 400 ng/mg Cr at the mid-luteal-phase peak and paralleled plasma Po concentrations. This non-radiometric assay for iPdG permits the assessment of ovulation, luteal formation and function, and luteolysis in unprocessed urine samples from domestic mares. Data from a single zebra indicate this approach also will permit simplified and non-invasive longitudinal studies of ovarian function among a wide range of Equidae.     

Use of a monoclonal antibody to estrone-3-glucuronide in an enzyme-linked immunosorbent assay (ELISA)

A direct urinary ELISA for estrone-3-glucuronide has been produced following cloning and characterisation of a monoclonal antibody to the above estrogen metabolite. The ELISA follows our established pattern of absorbing a thyroglobulin conjugate, to which estrone-3-glucuronide has been coupled, to the wells of a microtitre plate using guanidine hydrochloride. A competition reaction between either standards/samples and the adsorbed hormone compete for antibody combining sites. The assay is completed by addition of an anti-mouse Ig-peroxidase complex and read at 492 nm following additions of O-phenylenediamine substrate in under 4 h. The correlation between urinary "total estradiol" and "total estrone and estradiol" is very good and, in conjunction with our ELISA for pregnanediol glucuronide, has allowed for the improved clinical management of infertile and subfertile women.     

Hormonal and behavioural detection of oestrus in blackbuck, Antilope cervicapra, and successful artificial insemination with fresh and frozen semen

Hormonal detection of urinary pregnanediol-3 alpha-glucuronide proved an effective method of monitoring the progress of oestrous cycles in the blackbuck; observation of sexual behaviour in a vasectomized male was, however, a more practical procedure. Good correlation was observed between the occurrence of minimal pregnanediol concentrations in females and the maximal behavioural response by the male. On the basis of intervals between periods of behaviourally detected oestrus, a mean cycle length of 16.9 days (+/- 0.62, s.e.m.) was derived from 12 cycles (4 animals). Eleven females were inseminated in this study, 8 with freshly collected semen and 3 with frozen semen; 6 calves were obtained, 1 after the use of frozen semen. Pregnancy was monitored by measurements of pregnanediol-3 alpha-glucuronide excretion and by ultrasound scanning. The mean interval between insemination and parturition was 183.3 days inclusive, ranging from 182 to 186 days.     

The metabolism of estradiol-17β by the pregnant guinea pig uterus in vitro

The in vitro metabolism of [³H estradiol-17β-by the uterus was studied in non-pregnant, prenant (day 30-term) and post-parturant guinea pigs. Following incubation of tissue sections for one hour is Krebs-Ringer phosphate buffer, five major metabolites could be extracted from the medium or tissue depending upon age of gestation: estrone-3-glucuronide, estrone-3-sulfate, estradiol-3-glucuronide and estradiol-3-sulfate. Both sulfated estrogens were detected at each age of gestation studied, whereas the glucuronides, mainly of estrone, were not detected until approximately day 50. Thereafter, as term (day 65–70) was approached, their percentage contribution to total radioactivity increased at the expense of estradiol and the sulfates. Following parturition, total metabolites of estradiol rapidly decreased, particularly the glucuronides. No conjugates were detected in uteri from nonpregnant guinea pigs. In addition, no conjugates were found in the pre-partum mouse, rat and hamster or in human endometrium obtained immediately after birth. The data suggest that, in the guinea pig, a biochemical factor in the termination of normal pregnancy is the control of tissue levels of active estrogen (estradiol) by conjugation with glucuronic acid.     

Ovarian function in premenopausal women affected by breast cancer: the measurement of glucuronoconjugate metabolites of 17 beta-estradiol and progesterone throughout one entire menstrual cycle

For many years, hypersecretion of estrogens has been suspected of being one of the major risk factors of breast cancer for premenopausal women. Seventeen premenopausal women, who had undergone lumpectomy because of breast cancer (T1a No Mo) 3 yr before entering the study, were compared to 9 normal women of similar age, parity and body weight. A chemiluminescent method was used for the determination of estrone-3-glucuronide (E1-3G) and pregnanediol-3-glucuronide (Pd-3G) in early morning urine samples collected for an entire menstrual cycle of each of the 26 subjects. During the follicular phase, no significant differences in E1-3G and/or Pd-3G excretion were found between the two groups. During the luteal phase the E1-3G/Pd-3G ratio in the early, middle and late luteal phase had significantly increased in the women with breast cancer, in spite of normal Pd-3G excretion. Therefore, the measurement of glucuronoconjugate metabolites of ovarian hormones in overnight urine might be conveniently applied to the study of ovarian function in subjects with breast cancer. Furthermore, the results of this study may indicate that an estrogen/progesterone imbalance is an additional risk factor for the premenopausal breast cancer patient.     

Characterisation of monoclonal antibodies raised against testosterone

Using the antigens testosterone-17 beta-hemisuccinate and testosterone-3-(o-carboxymethyl) oxime, each coupled to bovine serum albumin, we have produced 44 monoclonal antibodies to testosterone. Of the 17 monoclonal antibodies raised against the 17 beta-linked antigen 8 showed extremely low affinity for testosterone (Ka less than or equal to 8 X 10(7) M-1) and none had an affinity greater than 5 X 10(9) M-1. Of the 27 monoclonal antibodies raised against the 3-linked antigen 2 had affinities less than 8 X 10(7) M, 7 had affinities greater than 5 X 10(9) M-1 and one had an affinity (Ka = 9 X 10(10) M-1) greater than that of a high affinity rabbit antiserum (Ka = 6 X 10(10) M-1). The affinity constant (Ka = 5 X 10(9) M-1) measured in the serum of the mouse whose spleen gave rise to the greatest number of high affinity antibodies, was significantly higher than those measured in the sera of the remaining mice (Ka = 0.7 - 3 X 10(8) M-1). The cross-reactions of the monoclonal antibodies varied widely but none showed an overall improvement in specificity when compared with the corresponding rabbit antisera. Results suggest that as well as the structure of the steroid antigen careful selection of the spleen donor facilitates the development of monoclonal antibodies with good binding characteristics.     

Properties of quercetin conjugates: modulation of LDL oxidation and binding to human serum albumin

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3'-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2 microM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3'-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide > quercetin > quercetin-3-glucuronide = quercetin-3-glucoside > catechin > quercetin-4'-glucuronide > isorhamnetin-3-glucuronide > quercetin-3'-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3'-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3'-sulfate > quercetin-7-glucuronide > quercetin-3-glucuronide > quercetin-4'-glucuronide = isorahmnetin-3-glucuronide). Kq values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3'-sulfate (approximately 4 microM)= quercetin > or = quercetin-7-glucuronide > quercetin-3-glucuronide = quercetin-3-glucoside > isorhamnetin-3-glucuronide > quercetin-4'-glucuronide (approximately 20 microM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.     

Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C(3)]isoflavone internal standards

A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C(3)]daidzein and [13C(3)]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4(')-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing.     

Menstrual cycle characterization and artificial insemination in the black mangabey (Cercocebus aterrimus)

Concentrations of immunoreactive estrone conjugates, pregnanediol-3-glucuronide, and luteinizing hormone were measured and indexed to creatinine in daily urine samples from three female black mangabeys (Cercocebus aterrimus). Daily observations of menstruation and perineal tumescence were recorded. The mean ± SEM lengths of the menstrual cycle [apparent cycle length of 26.0 ± 0.8 days determined by observation of intermenstrual intervals (n = 26); physiologic cycle length of 31.3 ± 5 days determined by urinary endocrine analysis (n = 4)], follicular phase [16.5 ± 4 days (n = 4)], and luteal phase [14.8 ± 1 day (n = 4)] were determined. The apparent cycle length is probably more accurate. Perineal tumescence began during or shortly after menstruation, increased concomitantly with increasing follicular phase conjugated estrone values, and reached maximal size in the periovulatory period. Ovulation was closely followed by a drop in conjugated estrone levels, an increase in urinary pregnanediol-3-glucuronide, and perineal detumescence. Peak concentrations of conjugated estrone and luteinizing hormone values were coincident. Pregnanediol-3-glucuronide accurately reflected luteal function in the black mangabey. Knowledge of the menstrual cycle parameters and their correlation to perineal tumescence was used to time artificial inseminations. Semen was obtained by rectal electroejaculation. Coagulum and extended semen, or trypsin-digested coagulum, were used for insemination. One insemination of trypsin-digested coagulum at the external os of the cervix resulted in a probable conception, follówed by apparent abortion after 3 weeks.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.