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It has been suggested that it is possible to monitor the menstrual cycle by measuring the concentration of urinary reproductive steroids. This neglects the variation in void volume and in urine production rate. In neither case has any systematic analysis been reported previously. Overnight urine samples were collected each day for one complete cycle by 24 women and the void volumes and intervoid times were recorded. The void volume and urine production rate were approximately lognormally distributed and the intervoid time was approximately normally distributed. Using these distributions we consider the implications of the variation in void volume and urine production rate for the comparison of the concentrations of a urinary analyte in two samples. [ABSTRACT FROM AUTHOR]

An enzyme-linked immunosorbent assay (ELISA) for measurement of pregnanediol-3α-glucuronide (PdG) excretion rates in urine samples diluted to 150 mL/h before analysis is described. The sensitivity of the 9 optimized standard curves was 0.093 ± 0.070 μmol PdG/24 hr, with the multiple combined standard curves having a mean mid-point (EC50) of 6.88 μmol PdG/24 hr. The PdG threshold excretion rate of 7.0 μmol/24 hr, which is used as a marker for the end of fertility, was situated in the most accurate region of the standard curve. The specificity of the ELISA was determined using normal variate transformation to compare seven menstrual cycle profiles obtained with the ELISA method with the profiles obtained previously using a validated radioimmunoassay (RIA) method. The cycle profiles all agreed within experimental error, and a high degree of correlation using Deming regression was obtained. The correlation equation was Y = 1.57X-0.11 μmol PdG/24 hr (n = 200; r = 0.932). The PdG excretion rates determined by the ELISA were 50% higher than given by RIA, but the normal ranges were similar to those given by the original reference gas liquid chromatographic method. The ELISA assay was therefore suitable as a reference method for measurement of thresholds of PdG excretion rates. [ABSTRACT FROM PUBLISHER]

Study Question: Do the basal body temperature (BBT) shift and the cervical mucus markers for the beginning of the post-ovulatory infertile phase (POIP) of a menstrual cycle agree with the corresponding urinary pregnanediol glucuronide (PdG) threshold value?Summary Answer: Perfect agreement between the cervical mucus markers and BBT shift and the hormonal definition of the start of post-ovulatory infertility occurred for only 7-17% of the cycles.What Is Known Already: The PdG threshold of 7.0 µmol/24 h is an objective and accurate marker for the beginning of the POIP. The rise in serum progesterone also produces the BBT shift and changes in cervical mucus which determine the mucus peak. Serum progesterone and urinary PdG are closely correlated when variations in urine volume are taken into account.Study Design, Size, Duration: Individual menstrual cycle profiles of urinary PdG excretion rates for 91 fertile cycles from normally cycling women were analysed to identify the day of the beginning of the POIP. These days were compared with those determined by the day of the BBT shift +2 days, the day of the mucus peak +4 days and the later of these two indicators. The study lasted 3 years.Participants/materials, Setting, Methods: A total of 62 women with normal menstrual cycles were recruited from three centres: Palmerston North, New Zealand; Sydney, Australia and Santiago, Chile. The cycles were displayed individually in a proprietary database program which recorded the PdG excretion rates, the BBT shift day and the cervical mucus peak day. A group of 15 women from a separate Chilean study had PdG urinary data measured as well as their day of ovulation determined by ultrasound.Main Results and the Role Of Chance: The BBT and cervical mucus markers differed significantly in their identification of the beginning of the POIP when compared with the PdG excretion rate of 7.0 µmol/24 h. The observation that the BBT shift day and the mucus peak day could be identified even though the PdG excretion rates were still at baseline levels in some cycles could lead to an unexpected pregnancy for women using these natural family planning (NFP) indicators.Limitations, Reasons For Caution: The study consisted only of fertile cycles from women with regular cycles of 20-40 days duration. All the women were intending to avoid a pregnancy during the study, thus the limits of the fertile window were not tested.Wider Implications Of the Findings: The NFP signals occurring earlier than the PdG threshold day could lead to an unexpected pregnancy. The signals occurring on the same day or later than the PdG threshold would not lead to unexpected pregnancies, but would require extra abstinence that could lead to non-compliance with the NFP method. A possible improvement in reliability of NFP methods is suggested.Study Funding/competing Interests: This study (project #90905) was funded by the NDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP). D.G.C. currently works for a diagnostic development company, Science Haven Ltd. The other authors have nothing to declare. [ABSTRACT FROM AUTHOR]

Ligand-binding assays, such as immunoassays, are usually analysed using standard curves based on the four-parameter and five-parameter logistic models. An estimate of the uncertainty of an analyte concentration obtained from such curves is needed for confidence intervals or precision profiles. Using a numerical simulation approach, it is shown that the uncertainty of the analyte concentration estimate becomes significant at the extremes of the concentration range and that this is affected significantly by the steepness of the standard curve. We also provide expressions for the coefficient of variation of the analyte concentration estimate from which confidence intervals and the precision profile can be obtained. Using three examples, we show that the expressions perform well. [ABSTRACT FROM AUTHOR]

Human milk lysozyme was conjugated with estrone glucuronide to give a monoacylated conjugate, two disubstituted isoforms, and one trisubstituted isoform in 99.4% yield. The conjugates were pure and highly inhibited (>98%) by the anti-estrone glucuronide antibody. The clearing curves were biphasic for all four conjugates but a 3 min initial rate assay was established and used to measure a normal menstrual cycle profile of estrone glucuronide excretion rates. The marked differences between the hen egg white and human milk lysozyme conjugates show that near identical tertiary structures do not necessarily imply similar physical, chemical, biochemical, and kinetic behavior. [ABSTRACT FROM PUBLISHER]

STUDY QUESTION What are the characteristics of, and how variable are, individual normal menstrual cycle profiles of excretion rates for the urinary metabolites oestrone glucuronide (E1G) and pregnanediol glucuronide (PdG)? SUMMARY ANSWER There is a continuum of menstrual cycle profiles that differ from standard textbook profiles but which can be understood simply in terms of growth, atresia and ovulation of ovarian follicles. WHAT IS KNOWN ALREADY Point-of-care assays with the Ovarian Monitor pre-coated assay tubes, using urine samples diluted to a constant volume per unit time, give laboratory accurate clinical data for individual menstrual cycles. Lay operators can perform the point-of-care assay system at home to achieve reliable and reproducible results, which can be used for natural family planning. STUDY DESIGN, SIZE, DURATION This prospective study involved 62 women, with normal menstrual cycles, recruited from three centres: Palmerston North, New Zealand, Sydney, Australia and Santiago, Chile. The study lasted 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Women collected daily urine samples and determined their E1G and PdG rates with a pre-coated enzyme assay system known as the Ovarian Monitor. For two cycles, the assays were repeated in a study centre and the results were averaged to give 113 individual menstrual cycles for analysis. The cycles were displayed individually in a proprietary database program. MAIN RESULTS AND THE ROLE OF CHANCE The individual normal hormonal profiles were more complex than the classic composite curves for 40% of the cycles. Of 113 ostensibly normal cycles, only 91 were potentially fertile and 22 had some luteal phase defect. The oestrone glucuronide and PdG excretion rates were reliable and informative in the non-invasive elucidation of ovulation and ovarian function for both simple and complex profiles. Daily monitoring revealed the variability of normal menstrual cycle profiles. The LH peaks were variable and ambiguous markers for ovulation. LIMITATIONS, REASONS FOR CAUTION The study consisted of cycles only from women with regular cycles of 20–40 days duration. All the women were intending to avoid a pregnancy during the study thus the limits of the fertile window were not tested. WIDER IMPLICATIONS OF THE FINDINGS The principles established in this study should apply to cycles of any length. All peaks in oestrone glucuronide excretion should be tested by concurrent measurements of PdG, which gives a positive indication of the fate of the follicle it represents. The Ovarian Monitor provides a useful addition for practitioners of natural family planning. STUDY FUNDING/COMPETING INTEREST(S) Financial support for this study was obtained from the UNDP/UNFPA/World Bank/WHO Special Programme of Research, Development and Research Training in Human Reproduction (HRP). D.G.C. is currently employed by and holds stock in Manawatu Diagnostics Ltd, a company in the development phase of a potentially competing product. The remaining authors have nothing to declare. [ABSTRACT FROM PUBLISHER]

BACKGROUND The UNDP/WHO/World Bank/Special Programme of Research, Development and Research Training in Human Reproduction (Geneva) set up a study to determine whether it is feasible for women to monitor their ovarian activity reliably by home testing. Daily self-monitoring of urinary hormone metabolites for menstrual cycle assessment was evaluated by comparison of results obtained with the Home Ovarian Monitor by untrained users both at home and in study centres. METHODS Women collected daily data for urinary estrone glucuronide (E1G) and pregnanediol glucuronide (PdG) for two cycles, then the procedure was repeated in the women's local centre (in Chile, Australia or New Zealand) giving a total of 113 duplicate cycles. The tests were performed without the benefit of replicates or quality controls. The home and centre cycles were normalized and compared to identify assay errors, and the resulting home and centre menstrual cycle profiles were averaged. RESULTS Reliable mean cycle profiles were obtained with the home and centre excretion rates agreeing to within 36 ± 21 nmol/24 h for E1G and 0.77 ± 0.28 µmol/24 h for baseline PdG values (1–5 µmol/24 h). The cycles had a mean length of 28.1 ± 3.1 days (n = 112; 5th and 95th percentiles: 24 and 35 days, respectively), a mean follicular phase of 14.8 ± 3.1 days (n = 107; 5th and 95th percentiles: 11 and 21 days) and a mean luteal phase length of 13.3 ± 1.5 days (n = 106; 5th and 95th percentiles: 11 and 17 days), calculated from the day of the LH peak. CONCLUSIONS The study confirmed that the Ovarian Monitor pre-coated assay tubes worked well even in the hands of lay users, without standard curves, quality controls or replicates. Point-of-care monitoring to give reliable fertility data is feasible. [ABSTRACT FROM PUBLISHER]

Three structurally characterized estrone glucuronide-lysozyme conjugates, E1 (a 60:40 mixture acylated at K3 and K97), E3 (acylated at K33), and E5 (acylated at both K33 and K97) were isolated and purified using a combination of cation-exchange chromatography on S-sepaharose in 7 M urea and hydrophobic interaction chromatography on butyl sepharose. Urea was essential to separate the conjugates into six chromatographically homogeneous fractions. In the absence of urea, complex mixtures of lysozyme and the six conjugate fractions were always encountered. The E1, E3, and E5 conjugates were highly inhibited by a sheep polyclonal anti-estrone glucuronide antibody only after the hydrophobic interaction chromatography step. The high level of inhibition enabled all three conjugates to be utilized as signal generators in homogenous enzyme immunoassays for urinary estrone glucuronide. Despite the apparently higher affinity of E3 for the antibody, both E1 and E3 gave standard curves that were indistinguishable provided that 1.7-fold more antiserum was used for E1. Both E1 and E3 yielded menstrual cycle urinary data that agreed with that provided by the Ovarian Monitor pre-coated assay tubes. Although, the menstrual cycle pattern was similar for the three signal generators, the E1G excretion rates yielded by E5 as the signal generator were only 60% of the reference values. Despite structural differences, there was no advantage gained in separating E1 and E3, but higher substituted conjugates such as E5 need removal for best assay performance. [ABSTRACT FROM AUTHOR]

1. Sheep liver cytoplasmic aldehyde dehydrogenase showed little pH dependence of V or kcat. Some buffer anion effects were noted. 2. The oxidation of aldehydes at pH 7.6 was quantitative but irreversible. The initial velocity data indicated a sequential mechanism for the addition of substrates. Inhibition by NADH and the product analogue 2-bromo-2-phenylacetic acid, together with the known tight binding of NADH to the free enzyme, indicated an ordered mechanism with NAD+ as leading substrate. 3. Values for the rate of binding and dissociation of NAD+ were obtained from the steady-state data. The values obtained were virtually identical with those which could be calculated from the data for the horse liver cytoplasmic enzyme. Close similarities are in general apparent for the horse and sheep liver cytoplasmic enzymes and with other tissue aldehyde dehydrogenases. 4. Apparent substrate activation was observed with high concentrations of both acetaldehyde and propionaldehyde, a limiting value of 0.25s-1 being obtained for Kcat No isotope effect was observed on V using [1-²H]propionaldehyde as substrate suggesting that NADH release might be rate-limiting in the steady-state. 5. The implications of the non-linear steady-state behaviour are discussed. [ABSTRACT FROM AUTHOR]

The oxidation of a series of primary alcohols by liver alcohol dehydrogenase has been studied under conditions of [S]0 [This symbol cannot be presented in ASCII format] [E]0 using the stopped-flow method. A biphasic process, with exponential rise to a steady state, was observed for most of the alcohols and the rate constants for the transient phase were determined. No transient phase could be detected for 2-chloroethanol and 2-nitroethanol and steady-state measurements were made for these alcohols. The rate constants for the hydrogen transfer step were obtained from the pre-steady-state rate constants for the various alcohols and correlated with the Taft σ* constant. The [This symbol cannot be presented in ASCII format]* value obtained (-1.8) is consistent with rate-limiting hydride transfer coupled with removal of the hydroxyl proton by a suitable basic group on the enzyme. A possible identity for this group is suggested. [ABSTRACT FROM AUTHOR]

1. The oxidation of benzyl alcohols by liver alcohol dehydrogenase under conditions [S]o » [E]o is a biphasic process with exponential rise to a steady state. The rate constants for the transient phase were determined by a curve-fitting procedure. 2. A large isotope effect was obtained for p-methoxybenzyl alcohol, indicating that hydrogen transfer is rate-determining for the transient phase. Rate constants for the hydrogen transfer step, k3, were obtained for the various alcohols and correlated with the Hammett σ constant by plotting log k3 against σ. The slope of this line (q = -0.76) is consistent with rate-limiting hydride transfer; a mechanism is proposed to account for the low magnitude of q. 3. The pre-steady-state rate constants, kb, for ali benzyl alcohols (<20 s -1) are lower than that for ethanol. 2-Methylpropan-l-ol and cyclohexylmethanol have pre-steady-state rate constants of about 150 s--1, indicating that the lower kb values for benzyl alcohols are due to the electronwithdrawing effect of the benzene ring, rather than to steric effects. [ABSTRACT FROM AUTHOR]

1. Cytoplasmic aldehyde dehydrogenase was shown to be free of contamination by the Mitochondrial enzyme by isoelectric focusing. 2. Both enzymes showed multiple banding in activity stains. The cytoplasmic enzyme gave two very close bands pI = 5.22 ± 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pI = 5.22 and five further bands of pI 5.48 ± 0.09, 5.56 ± 0.07, 5.65 ± 0.06, 5.70 ± 0.03 and 5.76 ± 0.02. Possible origins of the isoenzymes are discussed. 3. Disulfiram in a fourfold excess reduced the activity of the cytoplasmic enzyme to 9% of the initial value. The residual activity represents the activity of the disulfiram-modified enzyme and is not due to mitochondrial contamination. This casts doubt on the role of an essential thiol group. 4. The mitochondrial enzyme shows a low amplitude (22%) burst in the production of 4-nitrophenoxide ion during the hydrolysis of 4-nitrophenyl acetate at pH 7.6. The burst rate constant was 7.3 ± 1 s-1 and the steady-state rate constant was 0.2 s-1, values similar to those previously reported for the cytoplasmic enzyme. 5. The mitochondrial enzyme shows a burst in the release of protons during the oxidation of propionaldehyde at pH 7.6. The burst rate constant was 6 s-1 and the amplitude was equal to half the formal enzyme concentration. The significance of these results for the steady-state mechanism is discussed. [ABSTRACT FROM AUTHOR]

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with specific activity of 380 nmol NADH min-1 mg-1. An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions. On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6-fold enhancement of nucleotide fluorescence. The protein fluorescence (λexcit = 290 nm, λemission = 340 nm) is quenched on the binding of NAD+ and NADPH. The enhancement of nucleotide fluorescence on the binding of NADH has been utilized to determine the dissociation constant for the enzyme · NADH complex (Kd = 1.2 ± 0.2 μM). A Hill plot of the data gave a straight line with a slope of 1.0 ± 0.3 indicating the absence of co-operative effects. Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecylsulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme. Molecular weight were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212000 ± 8000 and 205000 respectively. Each enzyme consisted of four subunits with molecular weight of 53000 ± 2000. Properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases. [ABSTRACT FROM AUTHOR]