Search

Practical domestic monitoring of the menstrual cycle requires measurements of urinary metabolites of reproductive hormones: oestrone glucuronide (E1G) and pregnanediol glucuronide (PdG). Data reported in the literature are expressed as (i) concentration, without or with either creatinine- or specific gravity correction, or (ii) excretion rates. This variation in such a fundamental issue prompts consideration of the relationships between the four measures. Because the menstrual cycle kinetics of E1G and PdG are complex, we consider measurements of urinary creatinine, urea, galactose, xylose and inulin which tend to be more stable. We show that uncorrected concentration measurements of these urinary analytes can be positively correlated, negatively correlated or uncorrelated with the serum concentration. Based on measurements of urinary creatinine concentrations, urinary specific gravity and creatinine excretion rates, we conclude that urinary analyte concentration are likely to be more reliable when creatinine-corrected rather than corrected using specific gravity, but that both are less reliable than measurements of the excretion rate. This has implications for the quantitation of any urinary analyte, but especially for the monitoring of the menstrual cycle in which changes in E1G and PdG from one day to the next can be physiologically significant for a woman monitoring her fertility.

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]

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]