Correlation between morning urine estriol concentration and 24-hour estriol excretion

In an attempt to circumvent the need for 24-hour urine collections for estriol analyses in assessment of fetal status, the possibility of using morning urine samples was investigated. Results indicate 1) good correlation between 24-hour estriol excretion and morning estriol concentration, and between corresponding E/C ratios, 2) similar morning and 24-hour estriol concentrations, 3) high dependence of estriol and creatinine excretion on 24-hour urine volume but not on morning volume, 4) larger variations in 24-hour than in morning urine volume, and 5) better consistency of values in morning than in 24-hour samples in pathologic pregnancy. The use of serial morning urine concentrations at least as an outpatient monitoring procedure is suggested.     

Rapid radioimmunoassay of total urinary estriol

We describe a radioimmunoassay for total urinary estriol in pregnancy. A 25-mul aliquot of the urine specimen is acid hydrolyzed, neutralized, and diluted before assay. We use rabbit antisera against estriol-6-(O-carboxymethyl)oxime/bovine serum albumin and ammonium sulfate precipitation at room temperature. Results are unaffected by glucose or methenamine mandelate, a urinary tract antiseptic. Using semi-automatic pipetting equipment, one laboratory technologist can complete 50 assays within 8 h. This technique is both reliable and convenient and should decrease the expense of routine estriol assays.     

Changes in estriol excretion in the urine and kidney function during Partusisten infusion

In a random controlled trial the effects of a betamimetic infusion (Fenoterol 2 micrograms/min) on the estriol excretion and the renal function were analysed in 30 patients in the 28.-34. weeks of pregnancy. It was stated that the estriol excretion in urine during the Partusisten infusion significant decreases (from 663 micrograms/2 h to 376 micrograms/2 h, i.e. to 56%). The diuresis and the creatinine-clearance showed a significant diminishing too (from 218 ml/2 h to 154 ml/2 h and from 131 ml/min to 88 ml/min). The decrease of estriol excretion is caused on the one hand by the diminution of GFR, on the other by a direct effect of betamimetics on the kidney. From the results obtained in this study the following consequences can be drawn: 1. The determination of urinary estriol during tocolytic treatment is not a suitable method for the monitoring the fetoplacental unit. 2. Applying the tocolysis in an infusion a possible reduction of water intake and a strict control of water balance is of great importance. 3. The betamimetic therapy in patients having an impaired renal function can be applied only with great precaution.     

Evaluation of a radioimmunoassay for serum unconjugated estriol using commercial reagents

A radioimmunoassay using a commercially available antiserum was evaluated for measurement of serum unconjugated estriol in pregnancy. The evaluation showed an inter-assay variance of 12.1%, intra-assay variance of 6.8%, sensitivity of 0.2-0.6 ng/ml (0.7-2.1 nmol/l), and average recovery of 85.3%. The assay is specific for unconjugated estriol, showing less than 1% cross-reactivity with estriol-3-sulfate and estriol-16-glucuronide. Normal limits were established from 7 to 40 weeks' gestation using 175 serum samples. No diurnal variation could be demonstrated at 8 a.m. and 3 p.m. Eighty-nine serum specimens and 82 urine specimens obtained from 18 high-risk pregnancies were within normal limits except in cases of intruterine fetal death, pre-eclampsia, and suspected placental sulfatase deficiency. Serial urinary estriol levels fluctuated as much as 75%, while serial serum samples varied by only 30%.     

Shared reaction in solid-phase immunoassay for estriol determination

In the search of factors responsible for the experimental difficulties in developing accurate and sensitive solid-phase immunoassay of steroids, an experimental model has been set up for the study of nonspecific interaction of the steroid analyte with the coating protein. Along with the development of a highly sensitive enzyme-linked, solid-phase immunoassay for estriol measurement, we observed evidence of shared reactions. This property, to our knowledge not previously described for monomeric, low-molecular-weight antigens like estrogens, has been attributed to the presence of bovine serum albumin, which is capable of binding estrogens through hydrophobic interactions. The addition of estriol in solution in large excess did not reach a complete inhibition of the binding, so the possibility was excluded that the antibody simply binds to the adsorbed estrogen. The simplest explanation for the occurrence of the reaction is the hypothesis that a family of antigen determinants arises when the estriol is conjugated to a protein carrier. The corresponding antibodies are revealed only when the estrogen participates to the actual analytical system in the form of a steroid-protein conjugate. In the experiment, the estriol has been recognized as being coupled with one or more amino acid side chains present around its site of covalent linkage to the immunogen protein. The discussed results may be of help in developing a solid-phase immunoassay of small antigens as steroids, but also in applying the hybridoma and phage display technologies, the screening methods of which are based on sensitized solid phases.     

Trial of intravenous therapy in women with low urinary estriol excretion

Estriol excretion in pregnancy is favourably improved following administration of 25% dextrose to patients with persistently low estriol excretion. A double-blind controlled trial was undertaken in 60 patients to assess the efficacy of other regimens of infusion therapy with Hartmann's solution, aminofusin, 10% dextrose, or ritodrine in Hartmann's solution. Estriol excretion rose above the lower limit of normal in 69% of the patients treated. There was no significant difference in success rates between the four solutions studied when subjected to analyses of variance and covariance. Fetal and placental weights were directly related to estriol excretion. Influences of the various therapeutic regimens on metabolic acidosis have been considered and possible reasons for therapeutic success discussed.     

The origin of the hydroxyl radical oxygen in the Fenton reaction

There is an ongoing discussion in the chemical literature regarding the nature of the highly reactive hydroxyl radical formed from the reaction between ferrous iron and hydrogen peroxide (the Fenton reaction). However, the fundamental experiment of directly determining the source of the hydroxyl radicals formed in the reaction has not yet been carried out. In this study, we have used both hydrogen peroxide and water labeled with 17O, together with ESR spin trapping, to detect the hydroxyl radicals formed in the reaction. ESR experiments were run in phosphate buffer with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, and either H2O2 or H2O labeled with 17O. The hydroxyl radical was generated by addition of Fe2+ ion to H2O2, or as a control, by photolysis of H2O2 in the ESR cavity. Observed ESR spectra were the sum of DMPO/.16OH and DMPO/.17OH radical adduct spectra. Within experimental accuracy, the percentage of 17O-labeled hydroxyl radical trapped by the DMPO was the same as in the original hydrogen peroxide, for either method of hydroxyl radical generation, indicating that the trapped hydroxyl radical was derived exclusively from hydrogen peroxide and that there was no exchange of oxygen atoms between H2O2 and solvent water. Likewise, the complementary reaction with ordinary H2O2 and 17O-labeled water also showed that none of the hydroxyl radical was derived from water. Our results do not preclude the ferryl intermediate, [Fe = O]2+ reacting with DMPO to form DMPO/.OH if the ferryl oxygen is derived from H2O2 rather than from a water ligand.     

Sister chromatid exchanges and cell division delays induced by diethylstilbestrol, estradiol, and estriol in human lymphocytes

It had been found previously that exposure of human lymphocytes in vitro to diethylstilbestrol (DES), a synthetic estrogen and known human carcinogen, led to the induction of sister chromatid exchanges. More sister chromatid exchanges were induced in cells from pregnant women than from men. To see if the effects of DES could be induced by other estrogens, lymphocytes from a man and a pregnant woman were treated in vitro with the natural estrogens estradiol and estriol. These did not induce sister chromatid exchanges. To see if the presence of exogenous female hormones might be responsible for the increase in DES-induced sister chromatid exchanges seen in cells from pregnant women, lymphocytes from a man and a pregnant woman were also treated in vitro simultaneously with DES, estradiol, estriol, and progesterone. Treatments with these exogenous hormones did not alter the number of DES-induced sister chromatid exchanges. Our previous studies showed that DES also inhibits in vitro proliferation of lymphocytes. The results reported here show that estradiol also strongly inhibits this proliferation but that estriol is only a weak inhibitor. The cause of delayed cell proliferation induced by DES and estradiol was 2-fold: some of the cells were delayed in phytohemagglutinin-mediated blast cell transformation but, additionally, most cells had a prolonged cell cycle because of an extended G2 phase. These studies also showed that DES, but not estradiol or estriol, induced a low level of polyploidy in human lymphocytes.     

1H MAS and 1H[23Na] double resonance NMR studies on the modification of surface hydroxyl groups of gamma-alumina by sodium

The modification of surface hydroxyl groups with sodium in a series of Na2CO3-gamma-Al2O3 catalysts was investigated as a function of both the Na2CO3 loading and the calcination temperature by means of 1H magic angle spinning (MAS) and 1H(23Na) spin-echo double resonance NMR techniques. The 1H NMR experiments revealed that sodium ions are homogeneously distributed over the alumina surface and closely coordinated with the surface hydroxyl groups. In the catalysts calcined at 250 degrees C, the acidic hydroxyl groups (with a chemical shift of 2.0 ppm) are preferentially associated with sodium ions at low Na2CO3 coverages (5 and 10%), while both the acidic and the basic (0 ppm) hydroxyl groups are accessible for sodium ions at high coverages (15 and 20%). The coordination causes a low-field shift of about 2 ppm in the 1H MAS spectra, and a broad signal at 4.5 ppm appears. It is interesting that the 4.5 ppm signal is completely suppressed in the 1H(23Na) MAS experiments, providing direct evidence that a strong interaction exists between adsorbed sodium ions and the surface hydroxyl groups. Increasing the calcination temperature to 450 degrees C results in preferential removal of the acidic hydroxyl groups, and only the most basic hydroxyl groups remain when the calcination temperature is raised to 600 degrees C. This is attributed to the formation of the coordinated species. [formula: see text] which enhances the acidity of the surface hydroxyl groups and prompts their dehydroxylation, especially at high calcination temperature. Correlation of the 1H MAS NMR results and catalytic activity measurements indicates that the basic hydroxyl groups are essential for the carbonyl sulfide hydrolysis reaction.     

Undetectable maternal serum unconjugated estriol levels in the second trimester: risk of perinatal complications associated with placental sulfatase deficiency

OBJECTIVE: Our purpose was to determine the prevalence of undetectably low second-trimester maternal serum unconjugated estriol levels and the association with increased perinatal morbidity or mortality in pregnancies at risk for placental sulfatase deficiency. STUDY DESIGN: Nine centers in New England identified singleton pregnancies with undetectably low unconjugated estriol levels. Each unexplained case was matched with four controls; pregnancy outcome information was sought. RESULTS: Among 130,295 pregnancies surveyed, undetectably low unconjugated estriol levels were identified in 167 (13/10,000). Explanations included fetal death (53), overestimated gestational age (50), nonpregnancy (12), and chromosome abnormalities (5). The 41 unexplained cases were compared with 163 matched controls. Male offspring were more frequent (85%) among cases than among controls (55%). Although rates of perinatal complications were not significantly different, primary cesarean sections occurred about twice as often among cases. No perinatal deaths occurred. CONCLUSIONS: Neither severity of symptoms nor perinatal morbidity or mortality currently warrant routine interpretation of unexplained undetectably low unconjugated estriol levels as a marker for placental sulfatase deficiency.     

The prognostic and diagnostic value of total estriol in urine and in serum and of human placental lactogen hormone in serum in the last part of pregnancy

The benefit to the antenatal care from using estriol and human placental lactogen (hPL) determinations is emphasized and likewise that special attention must be paid to values below the reference intervals. Referring to biosynthesis and place of production, a survey is given of the various causes of low estriol and/or low hPL values, and a number of clinical cases illustrate the matter. A follow-up study of children from pregnancies with low estriol values has disclosed an alarming number of cases of severe handicaps.     

The heme prosthetic group of lactoperoxidase. Structural characteristics of heme l and heme l-peptides

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme l, is isolated through an approach that combines proteolytic hydrolysis and reverse-phase high performance liquid chromatographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotrypsin) or a peptide-free heme (with proteinase K). Both heme l and heme l-peptide species were investigated by paramagnetic 1H NMR spectroscopy, electrospray mass spectrometry, and peptide sequence analysis. Paramagnetic 1H NMR experiments on the low spin bis(cyano)-Fe(III)heme l complex conclusively define the heme l structure as a 1,5-bis(hydroxymethyl) derivative of heme b. The electrospray mass spectrum of heme l confirms the two-site hydroxyl functionalization on this heme. Paramagnetic 1H NMR spectra of the high spin bis(dimethyl sulfoxide)-Fe(III) complexes of the isolated heme species provide information regarding peptide content. Sequence analyses of peptides released from two heme l-peptide species by base hydrolysis suggest that heme-protein ester linkages in lactoperoxidase occur between the two hydroxyl groups of heme l and the carboxylic side chains of glutamate 275 and aspartate 125. These results confirm the earlier reported structural proposal (Rae, T. D., and Goff, H. M. (1996) J. Am. Chem. Soc. 118, 2103-2104).     

Prevalence of vitamin D insufficiency in an adult normal population

Demethylation of some aconitine-type norditerpenoid alkaloids was carried out with trimethylsilyl iodide and with HBr in glacial AcOH. Aconitine (10), cammaconine (23), delphinine (3), falconerine (18), lappaconitine (22), and talatizamine (24) afforded partially demethylated products. When methoxyl groups are present at the C-16 and C-18 positions, these are demethylated, and the methoxyl group at the C-1 position underwent demethylation in none the alkaloids studied except falconerine (18). With HBr-AcOH, in the case of alkaloids possessing a C-3 hydroxyl group, the methoxymethyl at C-18 formed a tetrahydrofuran, cyclizing at the C-6 position. Detailed NMR spectral studies (1H, 13C, 1H homonuclear COSY, HETCOR, and selective INEPT) carried out on the demethylation products have enabled accurate chemical shift assignments to be made for the demethylated alkaloids.     

Hydrogen atom abstraction of 3,5-disubstituted analogues of paracetamol by horseradish peroxidase and cytochrome P450

1. The formation of free radicals during enzyme catalysed oxidation of eight 3,5-disubstituted analogues of paracetamol (PAR) has been studied. A simple peroxidase system as well as cytochrome P450-containing systems were used. Radicals were detected by electron spin resonance (ESR) on incubation of PAR and 3,5-diCH3-, 3,5-diC2H5-, 3,5-ditC4H9-, 3,5-diOCH3-, 3,5-diSCH3-, 3,5-diF-, 3,5-diCl- and 3,5-diBr-substituted analogues of PAR with horseradish peroxidase in the presence of hydrogen peroxide (H2O2). Initial analysis of the observed ESR spectra revealed all radical species to be phenoxy radicals, based on the absence of dominant nitrogen hyperfine splittings. No radicals were detected in rat liver cytochrome P450-containing microsomal or reconstituted systems. 2. To rationalize the observed ESR spectra, hydrogen atom abstraction of PAR and four of the 3,5-disubstituted analogues (3,5-diCH3-, 3,5-diOCH3-, 3,5-diF- and 3,5-diCl-PAR) was calculated using ab initio calculations, and a singlet oxygen atom was used as the oxidizing species. The calculations indicated that for all compounds studied an initial hydrogen atom abstraction from the phenolic hydroxyl group is favoured by approximately 125 kJ/mol over an initial hydrogen atom abstraction from the acetylamino nitrogen atom, and that after hydrogen abstraction from the phenolic hydroxyl group, the unpaired electron remains predominantly localised at the phenoxy oxygen atom (+/-85%). 3. The experimental finding of phenoxy radicals in horseradish peroxidase/H2O2 incubations paralleled these theoretical findings. The failure to detect experimentally phenoxy radicals in cytochrome P450-catalysed oxidation of any of the eight 3,5-disubstituted PAR analogues is more likely due to the reducing effects that agents like NADPH and protein thiol groups have on phenoxy radicals rather than on the physical instability of the respective substrate radicals.     

A comparative multicenter study of the effects of continuous low-dose estradiol released from a new vaginal ring versus estriol vaginal pessaries in postmenopausal women with symptoms and signs of urogenital atrophy

OBJECTIVE: Our purpose was to study the efficacy, safety, and acceptability of a new estradiol-releasing (6.5 to 9.5 micrograms per 24 hours) silicone rubber vaginal ring compared with Ovesterin 0.5 mg estriol vaginal pessaries. STUDY DESIGN: Gynecologic clinical status, vaginal pH, cytologic characteristics, and occurrence of bacteriuria were determined before starting and after 3 and 12 weeks of treatment in 146 postmenopausal women. RESULTS: Both treatments alleviated the subjective and objective symptoms of estrogen deficiency excellently, and both were equally effective at restoring the vaginal pH to levels normally seen in fertile women (< 5.5). Vaginal cytologic studies showed a significant difference in maturation value in favor of the estradiol-releasing silicone rubber vaginal ring, as measured by the pathologist's assessment of the proliferation of the vaginal mucosa. A total of 77% of users were classified as responders, compared with 39% in the pessary group. Both treatments were well accepted. The administration of the pessary was associated with a significantly higher (p < 0.001) incidence of discomfort than that of the ring, which was given better (p < 0.001) rating by the patients at the 12-week visit. A strong preference (p < 0.001) for the ring was shown by patients with previous experience with pessaries. CONCLUSION: Treatment of urogenital symptoms in postmenopausal women with an estradiol-releasing vaginal ring is shown to be an effective and safe method, exhibiting advantages over treatment with estriol vaginal pessaries.     

Characteristics of mass fragmentation of steroids by atmospheric pressure chemical ionization-mass spectrometry

The characteristics of the mass spectra of sixty steroids were investigated using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). In APCI-MS, the drift voltage and nebulizer temperature affected the appearance of molecular ions. Solvent adducted ions [M+H+CH3CN]+ and [M+H+H2O]+ were decreased with an increase in drift voltage. The optimum drift voltage differed slightly for each steroid. These sixty steroids were divided into two groups according to their mass spectra profiles: one having a carbonyl group at position 3 together with a double bond at position 4 (group A) and the other bearing a hydroxyl group at the 3 position (group B). In group A, the predominant peak observed corresponded to the protonated molecular ion [M+H]+. The fragment ion corresponding to the elimination of CH2OH, COCH3 and/or COCH2OH from the steroid skeleton appeared as a base peak in some steroids of group A. In group B, predominantly [M+H-H2O]+ and/or [M+H-2H2O]+ ion(s), originating from the loss of water molecules, were observed. Other major ions in this group were the protonated molecular ions. Like other chemical ionization mass spectrometry, the mass spectrum of each steroid was very simple. These results suggest that APCI-MS is a suitable tool for the determination of the mass number of polar, nonvolatile and thermolabile steroids without derivatization.     

Synthesis and Characterization of Rare Earth Complexes with Phthalanilic Acids and Amino Acids

The phthalanilic acid compounds and the.anuno acid compounds have been follnd to havewide application as drug and plant groWth stimulator. Their transition metal complexes haveexcellent biological activities. Supported byNorthwest Highland Biological Research institute of China Academy of Sciellces, we haveachieved remarkable increasing yield of winterwheat at Xining Farm for three years. Thedrixed ligand complexes of this preparation areprobably new kind of the plant growth regulator.1 Ex…     

Tandem mass spectrometry and nuclear magnetic resonance spectroscopy studies of Candida bombicola sophorolipids and product formed on hydrolysis by cutinase

Natural mixtures of sophorolipids produced by the yeast Candida bombicola have been analyzed by fast atom bombardment (FAB)-MS and collision-induced dissociation (CID)-MS. Some pure components have been analysed by two-dimensional NMR spectroscopy. The presence of acidic, lactonic, and O-acetylated forms and the position of double bonds in the fatty acid part of these glycolipids can be easily inferred from positive and negative ion FAB-mass spectra. Details about position of O-acetylation can be obtained from CID mass spectra of [M+H]+ and [M-H]- ions and from the NMR spectra. Differences in CID fragmentation between protonated and sodiated molecular ions are discussed in detail. Enzymatic hydrolysis of 6',6"-di-O-acetyl sophorolipid lactone by cutinase from Fusarium solani results specifically in the removal of the 6'-O-acetyl group, whereas the 6"-O-acetyl and lactone group are resistant. This specificity is explained from a three-dimensional model of the sophorolipid generated on the basis of the short 1H,1H distances as inferred from the NMR (ROESY) spectra.     

Molecular dynamics and conformation in the gel and liquid-crystalline phases of phosphatidylethanolamine bilayers

Solid-state deuterium and carbon-13 nuclear magnetic resonance (NMR) spectra have been used to study the molecular dynamics and conformation of dipalmitoylphosphatidylethanolamine (DPPE) in both the gel (L beta) and liquid-crystalline (L alpha) phases. For this purpose DPPE was labeled with 13C in the carbonyl group of the sn-2 chain and with 2H at three different positions--4, 8, and 12--of the sn-2 chain, at the 2 position of the glycerol backbone, and at the 1 position of the ethanolamine head group. The 13C carbonyl and 2H chain spectra indicate that in the gel phase the DPPE molecules are diffusing about their long axes at rates of 10(5)-10(6) s-1 and the acyl chains are in an approximately all-trans conformation. The glycerol backbone spectra suggest that the backbone is in a gauche conformation in the gel state, rather than a trans conformation such as found in single crystals. The head group spectra in the gel phase are broad, featureless lines of about 20-kHz width. At the L beta leads to L alpha phase transition several changes take place. As is well-known, the chains disorder, and fast long-axis rotational diffusion begins, which results in the sharp, axially symmetric L alpha phase 2H spectra, which are a factor of 2 narrower than those observed in the L beta phase. The head group spectra also sharpen substantially at the transition, although their total width remains approximately constant. The invariance of the spectral width suggests that the average head group conformation is similar in both phases. However, the sharper spectra seen in the L alpha phase indicate that the rates of the head group motions in this phase are at least 3 orders of magnitude faster than those in the L beta phase. Thirdly, the 2H spectra of the glycerol backbone labeled DPPE narrow by a factor of about 4, and we believe this is due to a conformational change in this region of the molecule. Consistent with this interpretation is the fact that the powder pattern exhibited by the sn-2 13C = O in the L beta phase collapses to an isotropic-like line at the phase transition.