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.     

Oestrogen conjugates of human late-pregnancy urine

1. The separation of the oestrogen conjugates in late-pregnancy urine into two groups, peaks I and II, by gel filtration on Sephadex G-25 (Beling, 1963) has been shown to be affected by the presence of urate, which delays the elution of peak II conjugates. 2. By reapplication to a Sephadex column, peak I conjugates have been further separated into two groups (peaks IA and IB) and the metabolites in urine effecting this separation have been studied. 3. Further analysis of the mixed conjugates in the main groups IA, IB and II by ion-exchange and partition chromatography has led to the identification of some of the conjugates present. 4. Oestriol 3-sulphate 16α-glucuronide and 16α-hydroxyoestrone 3-sulphate 16α-glucuronide have been identified in peak IA. 5. The main components of peak IB have been identified as oestrone 3-glucuronide and oestriol 3-glucuronide. 6. The major conjugate in peak II was oestriol 16α-glucuronide and no oestriol 17β-glucuronide was found; small amounts of the ring-d monoglucuronides oestradiol 17β-glucuronide, 16-epioestriol 16β-glucuronide and 16α-hydroxyoestrone 16α-glucuronide were found in this fraction. 7. The behaviour of synthetic oestrogen monoglucuronides has been used as a guide in separation.     

Conjugation position of quercetin glucuronides and effect on biological activity

Quercetin glycosides are common dietary antioxidants. In general, however, potential biological effects of the circulating plasma metabolites (e.g., glucuronide conjugates) have not been measured. We have determined the rate of glucuronidation of quercetin at each position on the polyphenol ring by human liver cell-free extracts containing UDP-glucuronosyltransferases. The apparent affinity of UDP-glucuronosyltransferase followed the order 4′- > 3′- > 7- > 3, although the apparent maximum rate of formation was for the 7-position. The 5-position did not appear to be a site for conjugation. After isolation of individual glucuronides, the inhibition of xanthine oxidase and lipoxygenase were assessed. The K_i for the inhibition of xanthine oxidase by quercetin glucuronides followed the order 4′- > 3′- > 7- > 3-, with quercetin-4′-glucuronide a particularly potent inhibitor (K_i = 0.25 μM). The glucuronides, with the exception of quercetin-3-glucuronide, were also inhibitors of lipoxygenase. Quercetin glucuronides are metabolites of quercetin in humans, and these compounds can retain some biological activity depending on conjugation position at expected plasma concentrations.     

Studies on steroid conjugates: IX. Urinary excretion of sulfate conjugated metabolites of cortisol in man.

Following i.v. administration of [4-14C]cortisol, various sulfate conjugated metabolites of cortisol in urine were identified and their respective excretion rates measured. The results obtained demonstrated the following: 1) sulfate conjugates as a group are excreted considerably slower than glucuronide conjugates; 2) sulfate conjugates of steroids with non-reduced ring-A (C-21 sulfates) are excreted (and presumably formed) much faster than steroid-3-sulfates, which require reduction of the ring-A prior to the conjugation; 3) the excretion of C-3 sulfates of ring-A reduced steroids with glycerol side-chain (cortols and cortolones) is significantly faster than those of the corresponding steroids with dihydroxyacetone side-chain (THF, THE and their 5alpha-isomers); 4) the relative concentrations of C-21 sulfates of steroids with ring-A intact (FK, EK, ER, epiER and 6beta-hydroxycortisol) are much higher than the concentrations of C-21 glucuronides of these steroids.     

Formation of glucuronides of estradiol-17 beta by human mammary cancer cells

The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.     

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.     

Androgen glucuronides: I. Direct formation in rat accessory sex organs

A simple one-step procedure is described on the isolation of androgen glucuronides from various rat tissues. This procedure uses polyacrylamide gel electrophoresis, and permits a quantitative isolation of a single band containing the total androgen glucuronides without the contamination of free androgens and androgen sulfates. This procedure was used to determine the ability of various tissues of the rat to form androgen glucuronides directly when they were incubated with 1,2-[³H]-testosterone (0.1 μM) $\text{in}$$\text{vitro}$. Of eleven organs studied, only the accessory sex organs (ventral prostate, seminal vesicle, and coagulating gland), liver, and kidney were capable of forming androgen glucuronides. At the end of a one-hour incubation period, approximately 1% of the total radiolabeled steroids in the prostatic tissue minces were in the form of glucuronide conjugates. The predominant androgen glucuronide formed in the accessory sex organs was 5α-androstane-3α,17β-diol 17β-d-glucuronide. This is in contrast to the rat liver and kidney in which testosterone glucuronide was the predominant conjugate.A similar amount of labeled glucuronide conjugates was formed from either [³H]-testosterone, [³H]-dihydrotestosterone or [³H]-androstenedione, whereas negligible amount of steroid conjugates was formed from [³H]-cortisol. The formation of androgen glucuronides requires metabolically active tissues; furthermore, the conjugation process was inhibited by the antiandrogen, cyproterone acetate, or by metabolic inhibitors, such as oligomycin or N-ethylmaleimide.     

Flavonoid glucuronides from Picria fel-terrae

Three flavonoid glucuronides are reported from a n-BuOH extract of Picria fel-terrae (Scrophulariaceae). The structures were established by UV, one- and two-dimensional NMR and mass spectrometry as apigenin 7-O-β-glucuronide, luteolin 7-O-β-glucuronide and apigenin 7-O-β-(2″-O-α-rhamnosyl)glucuronide, the latter one being a new compound.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Separation of urinary oestrogen sulphates from oestrogen glucosiduronates on diethylaminoethyl-Sephadex columns

1. Gradient elution (sodium chloride concentration gradient) on DEAE-Sephadex columns is used to separate urinary oestrogen conjugates of pregnancy urine. Changes in the shape of the gradient alter the chromatograms in a predictable manner so that the dispersion of peaks can be modified at will. 2. Suitable choice of the gradient results in the separation from each other of oestrogen sulphates, oestrogen 16(or 17)-glucosiduronates, oestrogen 3-glucosiduronates and free oestrogens. 3. Evidence for the presence of oestriol 3-sulphate, oestrone 3-sulphate, 17β-oestradiol 3-sulphate, 16-oxo-17β-oestradiol 3-sulphate and oestriol 16(or 17)-sulphate in the sulphate fraction of DEAE-Sephadex chromatograms of pregnancy urine is provided.     

Conjugated and unconjugated plasma oestrogens in men with chronic alcoholism and in normal men

The plasma concentrations for unconjugated and conjugated oestrone, oestradiol-17β and oestriol, were measured in 25 male patients with chronic alcoholism and 25 healthy male blood donors of the same age. All persons had normal weight and were free of medication. The patients with chronic alcoholism consisted of 15 persons with and 10 without hepatomegaly. Liver biopsy was performed in the persons with hepatomegaly. Unconjugated oestrogens were separated on LH-20 micro columns after extraction with diethyl ether. Oestrogen sulphates were extracted and separated after hydrolysis with sulphatase, and glucuronides were extracted and separated after hydrolysis with glucuronidase.In the patients with hepatomegaly the plasma concentrations of unconjugated oestrone, oestradiol-17β and oestriol were significantly increased, and the plasma levels for the same oestrogen sulphates were significantly decreased, whereas a moderate increase was observed for oestriol glucuronide. The patients without hepatomegaly revealed a significantly lower plasma concentration for oestrone sulphate, whereas no difference was seen for the remaining oestrogen components. The ratio oestrone sulphate to oestrone was significantly reduced in both groups of patients. A significant decrease for the ratio oestradiol sulphate to oestradiol and oestriol sulphate to oestriol was seen only in the patients with hepatomegaly.     

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.     

Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.     

Glucuronidation of the mycotoxins alternariol and alternariol-9-methyl ether in vitro: chemical structures of glucuronides and activities of human UDP-glucuronosyltransferase isoforms

Alternariol (AOH) and alternariol-9-methyl ether (AME) are major toxins produced by fungi of the genus Alternaria and are frequently found in various food items. Because AOH has three hydroxyl groups and AME two, the formation of various glucuronides must be expected. When AOH was incubated with hepatic and intestinal microsomes from rats, pigs and humans in the presence of uridine diphosphate glucuronic acid, two glucuronides were detected and tentatively identified as AOH-3-O-glucuronide and AOH-9-O-glucuronide. Under the same conditions, AME yielded predominantly AME-3-O-glucuronide and only small amounts of AME-7-O-glucuronide. The activities of all microsomes for the glucuronidation of AOH and AME were in the same range. Nine out of ten recombinant human UDP-glucuronosyltransferases (UGTs) were able to glucuronidate AOH, and eight out of ten UGTs had activity for AME. These data suggest that AOH and AME are readily glucuronidated in hepatic and extrahepatic tissues, implying that glucuronidation constitutes a major metabolic pathway in the disposition of these mycotoxins. Presented at the Mycotoxin Workshop, Utrecht, Netherlands, April 28–30, 2008     

Microbial process for preparation of glucuronides of raloxifene

Raloxifene, also known as Evista^?, has recently been approved for the prevention of osteoporosis. Three glucuronidated compounds: raloxifene-6-glucuronide, raloxifene-27-glucuronide, and raloxifene-6,27-diglucuronide are known metabolites of raloxifene in man. Reference standards of the three glucuronides were needed for clinical trials. Although chemical routes exist to make the two mono-glucuronides, these routes were unable to provide material to meet the needs of clinical trial standards. No chemical route existed to synthesize the di-glucuronide. A bioconversion process using the microorganism Streptomyces sp NRRL 21489 was identified and scaled up. The biotranformation products were prepared in a tank fermentation, purified, and characterized by UV, LC/MS and NMR spectroscopy. Received 24 March 1999/ Accepted in revised form 26 June 1999     

Steroid glucuronides as male pheromones in the reproduction of the African catfish Clarias gariepinus--a brief review

After ovulation, female African catfish are strongly attracted by the odor of male conspecifics. This attraction depends on the presence of the seminal vesicle, a part of the male reproductive organs. Removal of the seminal vesicle illustrates this fact. A low dose of seminal vesicle fluid, added to the water, appears to be highly attractive for catfish which have ovulated. Fractionation of the fluid and testing of the different fractions shows that steroid glucuronides could be responsible for the attraction. These steroid glucuronides can be identified with gas chromatographic-mass spectrometric analysis. A mixture of glucuronides, prepared to resemble the composition of the seminal vesicle fluid, evokes a dose-dependent attraction. The most potent odorant, observed by measuring electrical responses from the olfactory epithelium and from the olfactory tract appears to be 3,17-dihydroxy-5β-pregnan-20-one-3-glucuronide.     

Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination

The development of an immunoaffinity-based extraction method for the determination of morphine and its glucuronides in human blood is described. For the preparation of an immunoadsorber, specific antisera (polyclonal, host: rabbit) against morphine, morphine-3-glucuronide and morphine-6-glucuronide were coupled to 1,1′-carbonyldiimidazole-activated trisacrylgel and used for immunoaffinity extraction of morphine and its glucuronides from coronary blood. The resulting extracts were analysed by HPLC with native fluorescence detection. The mean recoveries from spiked blood samples were 71%, 76% and 88% for morphine, morphine-3-glucuronide and morphine-6-glucuronide, respectively. The limit of detection was 3 ng/g blood and the limit of quantitation was 10 ng/g blood for all three analytes. The results of the analysis of coronary blood samples from 23 fatalities due to heroin are presented.     

Flavonoid glucuronides are substrates for human liver beta-glucuronidase

Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme beta-glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited beta-glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a beta-glucuronidase inhibitor). Furthermore, pure recombinant human beta-glucuronidase hydrolysed various flavonoid glucuronides, with a 20-fold variation in catalytic efficiency (k(cat)/K(m)=1.3x10(3) M(-1) s(-1) for equol-7-O-glucuronide and 26x10(3) M(-1) s(-1) for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal beta-glucuronidase from various human cells.     

Studies on the metabolism of oestrone and oestradiol-17 beta in the kidneys of minipigs of different ages and sexes (author's transl)]

After incubation of [4-14C]oestrone (E1) with kidney cortex slices of minipigs, [4-14C]oestradiol-17 beta (E2) and small amounts of a polar metabolite were detected in the ether-soluble fraction. E1, E2 and polar metabolites were found in the protein-bound fraction. The water-soluble fraction contained E1-3-glucuronide (80% of total glucuronides), E2-3-glucuronide and trace amounts of the 3-monoglucuronide of oestriol (E3). When E2 was used as substrate, the main product formed was E1; it was detected in both the ether-soluble and protein-bound fractions. E1-3-Glucuronide was the main metabolite in the water-soluble fraction, which also contained some E2-3-Glucuronide. In male minipigs, the mast, the rate of conversion of E1 and E2 as well as the formation of glucuronides were significantly greater in fertile females than in infertile females. Whereas no sex difference was observed in the metabolism of oestrogens in kidneys of infertile minipigs, the rate of oxidoreduction and glucuronidation was more pronounced in fertile female animals than in the corresponding males. The present results suggest that, in the kidneys of minipigs, the ratio of E1 to E2 is shifted towards the former; furthermore, by a comparatively rapid metabolism of the oestrogenic hormone, the renal tissue contributes to the maintenance of hormonal equilibrium.     

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.