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.     

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.     

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.     

Metabolites of estradiol-17β in guinea pig uterus late in pregnancy

The metabolism of estradiol-17β by the guinea pig uterus late in pregnancy was studied $\text{in vivo}$ and $\text{in vitro}$.Whole uteri were examined for estrogen metabolites one hour following an intravenous injection of [³H]-estradiol-17β or uterine sections were examined after incubation for one hour at 37°C in medium containing [³H]-estradiol-17β.In both instances uterine tissue metabolized estradiol-17g to five products: estrone, estrone-3-sulfate, 17β-estradiol-3-sulfate, estrone-3-glucuronide and 17β-estradiol-3-glucuronide. Of the total radioactive products 11 – 43% were glucuronides, 17 – 26% were sulfates and 4 – 17% was estrone. These results indicate that the guinea pig uterus actively transforms estradiol-17β into glucuronides and sulfates late in pregnancy.     

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.     

Specificity studies of monoclonal and rabbit antibodies raised against steroid glucuronides

Monoclonal and rabbit antibodies raised against estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide have been studied with respect to their ability to bind free estrone and its conjugates or free pregnanediol and its conjugates, respectively. High titre and high specificity were observed with monoclonal antibodies produced against pregnanediol-3 alpha-glucuronide, whereas the monoclonal antibodies produced against estrone-3-glucuronide were not so specific when compared with the corresponding rabbit antibodies. Both monoclonal and rabbit antibodies had affinity constants in the range of 10(9)--10(10) liter/mole.     

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.     

Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine

A hydrophilic interaction liquid chromatography-time-of-flight mass spectrometry (HILIC-TOFMS) method for the quantification and confirmation of morphine (M), codeine (C), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and codeine-6-glucuronide (C6G) is presented. The method was validated in terms of specificity, selectivity, extraction recovery, accuracy, repeatability, linearity and matrix effect. After a straightforward sample preparation by solid phase extraction (SPE) the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The HILIC technique provided good chromatographic separation which was critical for isomers M3G and M6G. The analytes were detected after electrospray ionization (ESI) in positive mode with mass accuracies below 2 mDa using a 5-mDa window. A measurement range of 50-5000 ng/ml was applied for calibration using deuterated analogs as internal standards. The precision of the method was 5.7% and 10.2% (RSD) within and between days, respectively. The applicability of the method was demonstrated with authentic urine samples known to contain codeine and/or morphine and their intact glucuronide conjugates. Identification of the analytes was based on in-source collision induced dissociation (ISCID), applying three diagnostic ions with accurate mass.     

Identification and synthesis of norhydromorphone, and determination of antinociceptive activities in the rat formalin test

The main objective of this paper is to report the identification and synthesis of norhydromorphone, a novel metabolite of hydromorphone, and its antinociceptive activities when tested in the formalin test as compared to other known analgesics. In addition, we are reporting for the first time the lack of antinociceptive activities of hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide in the rat formalin test. Norhydromorphone was isolated and identified as a metabolite of hydromorphone in a cancer patient's urine. An authentic standard of norhydromorphone was synthesized. The identity of norhydromorphone in the urine sample was confirmed by comparing the LC retention time and MS ion fragmentation with the synthetic standard using a liquid chromatographic-mass spectrometric-mass spectrometric (LC-MS-MS) assay. Norhydromorphone was found to be a minor metabolite of hydromorphone in the urine. Additionally, the antinociceptive activities of norhydromorphone, hydromorphone, morphine, dihydromorphine, dihydroisomorphine, hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide were determined in the rat formalin test following intraperitoneal (i.p.) administration. Only limited antinociception was observed and no significant increase in antinociception was detected at the three doses tested. The increased polarity of norhydromorphone as compared to hydromorphone due to the primary piperidine nitrogen may make it less favorable to cross the blood-brain-barrier (BBB), which may be partly responsible. In addition, lower intrinsic antinociceptive activity, which remains to be determined, could also contribute to the low antinociception. Our results also show that hydromorphone was five times as potent as morphine in the formalin test, while dihydromorphine and dihydroisomorphine were equipotent to and 36% as potent as morphine, respectively. Hydromorphone-3-glucuronide, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide did not exhibit any antinociceptive effect at the doses tested. The results further underscore the importance of a free C3-OH to the analgesic effect of morphine alkaloids.     

Evaluation of Urinary Protein Precipitation Protocols for the Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network

Standardization of sample collection, shipping, and storage has been a major focus of biorepositories servicing large, multi-institute studies. The standardization of total protein concentration measurements may also provide an important metric for characterizing biospecimens. The measurement of total protein concentration in urine is challenging because of widely variable sample dilutions obtained in the clinic and the lack of a reference matrix for use with a standard curve and blank subtraction. Urinary proteins are therefore typically precipitated and reconstituted in a reference solution before quantitation. We have tested three different methods for protein precipitation and evaluated them using variability in total protein concentration measurement as a metric. The methods were tested on four urine samples ranging from very concentrated to very dilute. A method using a commercially available kit provided the most reproducible results, with average coefficients of variation <10%. Addition of a freeze/thaw did not lead to significant protein loss or additional variability. Samples were titrated and the measurements obtained appeared to be linearly correlated with sample starting volume. This method was applied to analysis of 77 urine biorepository samples and provided reproducible results when the same sample was assayed on different microwell plates.     

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.     

Measurement of creatinine by Jaffe's reaction--determination of concentration of sodium hydroxide required for maximum color development in standard,urine and protein free filtrate of serum

Creatinine in serum or urine is determined by Jaffe's reaction where creatinine produces quantitatively an orange color with picric acid in alkaline medium. After allowing an incubation time of 15 min at room temperature for color development the color is measured at 520 nm. Without taking into consideration the acidic nature of standard, protein free filtrate (PFF) of serum and urine, 1% picric acid and 0.75N NaOH are used in this reaction for color development in standard, PFF of serum and urine. An investigation was thought to be necessary to determine the optimum alkali concentration required in standard, PFF of serum and urine. The results show that 0.25, 0.75 and 1N NaOH give maximum color in urine, standard and PFF of serum respectively. A standard solution of creatinine is prepared in 0.1N HCl and the PFF of serum is obtained by addition of fresh tungstic acid. Alkali is consumed to neutralise the acids in both these cases. For urine creatinine measurement, a direct diluted urine sample is used. The difference in the requirement of NaOH is conceivable. The routine use of 0.75N NaOH irrespective of the nature of specimen as is done in all biochemical laboratories, for creatinine measurement needs modification in the light of this investigation.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

The direct radioimmunoassay of oestrogen glucuronides in human female urine.

Radioimmunoassays for five oestrogen metabolites in urine are described; they are oestrone 3-glucuronide, oestradiol 3-glucuronide, oestradiol 17 beta-glucuronide, oestriol 3-glucuronide and oestriol 16 alpha-glucuronide. These assays have proved accurate and reliable and can be performed rapidly; they have been carried out directly in diluted menstrual cycle urine and pregnancy urine. No sample pretreatment was required. Preliminary results suggest that clinically useful information can be obtained by performing these assays on random urine specimens.     

Enzymatic detection of urinary conjugated steroids after gel chromatography

An enzymatic detection method is described for urinary conjugated steroids after chromatographic fractionation with Sephadex G-25. The principle of the method is as follows. Part of a 24-h urine sample, (1–2 ml of urine) is applied directly, to a short column of Sephadex G-25 and eluted with acetate buffer solution. Steroid conjugates in each fraction are hydrolyzed with steroid sulfatase—β-glucuronidase. After enzymatic hydrolysis, an enzymatic color development reagent for steroids, either 3α-hydroxysteroid dehydrogenase or 3β-hydroxysteroid oxidase, are added and the dye formed is measured spectrophotometrically. Excretion patterns of steroid-3β-sulfates, and steroid-3α-glucoronides and steroid-3α-sulfates are shown with some patients' samples. A precision of the assay values for steroid-3α-glucuronide, steroid-3α-sulfate and steroid-3β-sulfates in urine samples and assay values for normal subjects are also studied.This simple enzymatic method for detecting the excreption patterns of urinary conjugated steroids may have a diagnostic value for clinical tests.     

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.     

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.     

Determination of codeine and metabolites in plasma and urine using ion-pair high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method for the simultaneous determination of codeine and seven metabolites is described. The samples are purified by reversed-phase solid-phase extraction. Codeine, norcodeine, codeine-6-glucuronide, norcodeine-6-glucuronide and morphine-3-glucoronide are measured with UV detection. Detection limits are 3 nmol/l (morphine-3-glucuronide) to 20 nmol/l (codeine). Morphine, normorphine and morphine-6-glucuronide are measured with electrochemical detection. Detection limits are 0.4 nmol/l (morphine-6-glucuronide) to 1.0 nmol/l (normorphine). Correlation coefficients better than 0.998 are normally obtained for all compounds. The method was applied to the determination of the kinetics of codeine and its metabolites in plasma and urine samples from healthy volunteers.     

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of opiates and cocaine in meconium

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of 6-monoacetylmorphine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, cocaine, benzoylecgonine and cocaethylene in meconium using nalorfine as the internal standard. The analytes are initially extracted from the matrix by methanol (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or 0.01 M ammonium hydrogen carbonate buffer (morphine-3-glucuronide, morphine-6-glucuronide). Subsequently a solid-phase extraction with Bondelut Certify columns (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or ethyl solid-phase extraction columns (morphine-3-glucuronide, morphine-6-glucuronide) was applied. Chromatography was performed on a C(8) reversed-phase column using a gradient of acetic acid 1%-acetonitrile as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with atmospheric pressure ionisation-electrospray (ESI) interface. The method was validated in the range 0.005-1.00 microg/g using 1 g of meconium per assay and applied to analysis of meconium in newborns to assess fetal exposure to opiates and cocaine.     

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.