Thet complex of the mouse: Chemical characterization by urinary volatile profiles.

Urine samples from C3H congenic house mice (Mus domesticus) differing only at thet complex were examined by capillary gas chromatography to assess variations in the volatile components that may cause olfactory discrimination between animals bearingt lethal and+(wild-type) haplotypes. Urine was collected from 192 males and females varying in age from 1 to 9 months. C3H congenic mice that have the same genetic background at all loci but differed in theirt complex genotypes: +/ +, +/tw1,T/t w1, T/+ were used. No urinary volatiles were unique to thet complex. However, significant differences amongt complex genotypes and among ages occurred for concentrations of 12 male volatiles and four female volatiles. Usually young males (1-2 months of age) had significantly higher concentrations of cyclic enol ethers and ketones than older males (4-9 months of age). Moreover, some urinary volatiles (cyclic enol ethers, one ketone, dehydrobrevicomin, and thiazoline) were excreted in the urine of T/+and/orT/t males in significantly higher concentration than in the urine of +/+ males. Age andt complex genotype influences on the urinary volatiles in females were observed for four ketones. Gas chromatography of urinary components has the potential to be used in field studies of thet complex because the two t complex genotypes found in wild populations, +/+ and +/t, had significant differences in concentration for two males volatiles and three female volatiles.

Puberty-affecting synthetic analogs of urinary chemosignals in the house mouse, Mus domesticus.

Endocrinologically- and socially-dependent volatile constituents of female mouse urine, identified in a previous study, were tested for their capability to accelerate puberty and extend the estrous period in young females. Several volatile ketones advanced puberty by approximately three days and extended the period of vaginal cornification in 55-75% of exposed females. High High concentrations of these substances were capable of overriding the known puberty-delaying chemosignals. Volatile cyclic enol ethers were also effective in extending estrus, but not puberty acceleration.

Urinary volatile profiles of pine vole,Microtus pinetorum, and their endocrine dependency.

The volatile compounds identified by combined gas chromatography-mass spectrometry inMicrotus pinetorum urine include alcohols, aldehydes, hydrocarbons, ketones, nitriles, and pyrazines. Several lactone derivatives were found to be characteristic urinary substances of this species. Ovariectomy depressed concentrations of only five out of a great number of profile constituents. Elevating estrogen levels (by exposing females to male-soiled bedding or treating them with estradiol) tends to depress the urinary concentration of a number of selected volatiles. Estrogen implantation provoked a periodic increase in the level of three compounds (nonanal, benzal-dehyde, and an unidentified substance). The volatile profile of castrate male urine was similar to that of intact male urine. Female urine contained γ-octanoic lactone and two pyrazine derivatives in higher concentrations andp-methyI-propenylbenzene in a lower concentration, when compared to male urine. No qualitative differences between the urinary profiles of males and females were observed.

Separation of proteins by microcolumn liquid chromatography based on the reversed-phase and size-exclusion principles.

Slurry-packed fused-silica microcolumns of 250 micron I.D., are characterized for use in high-performance liquid chromatographic studies of proteins. The present work utilizes the reversed-phase and size-exclusion chromatographic modes for the separation of standard protein mixtures. A 5-micron, 300-A octyl material is utilized for the reversed-phase studies, and the size-exclusion studies are accomplished with 5-micron diol material of 60-, 100- and 300-A pore sizes. Column efficiency and packing reproducibility, as well as sample capacity in a micropreparative mode, are discussed. In addition, the inherent mass sensitivity of a microcolumn liquid chromatography system as applied to protein detection is demonstrated.

Dynamics of excretion of urinary chemosignals in the house mouse (Mus musculus) during the natural estrous cycle.

The volatile fraction of urinary metabolites was investigated chromatographically at five different stages of the natural estrous cycle. A very substantial endocrine dependency has been noted for 11 compounds: 4 ketones, 2 acetate esters, 3 dihydrofuran isomers, dehydro-exo-brevicomin, and 2,5-dimethylpyrazine. The compounds were structurally verified through combined gas chromatography/mass spectrometry.

Variations in mouse (Mus musculus) urinary volatiles during different periods of pregnancy and lactation.

Mouse urine samples from different pregnancy and lactation periods were examined by capillary gas chromatography to assess variations in the volatile signals that may affect the endocrine function of other females. Statistically significant changes in the excretion of certain urinary volatiles were observed; from 26 readily quantifiable constituents, 14 appear to be under the endocrine control. These selected components, positively identified through mass spectrometry and retention data, and the synthetic standards are ketones, unsaturated alcohols, esters, and cyclic vinyl ethers.

Estriol and its conjugates in late pregnancy determined by extraction with Carbopack B and liquid chromatography with fluorometric detection.

We report a method for measuring estriol and its intact conjugates in urine, serum, and amniotic fluid. A single assay can be done within about 50 min, eight samples assayed in less than 5 h. A 70-microL urine sample is diluted and the estriol conjugates are adsorbed from it onto graphitized carbon black (Carbopack B, Supelco). After two washings, the analytes are desorbed with chloroform/methanol (60/40, by vol) containing tetrapropylammonium bromide. After solvent evaporation, the residue is redissolved in 100 microL of water/acetonitrile and 20 microL is injected into the chromatograph. Or 1 mL of serum or 0.5 mL of amniotic fluid is deproteinized with cold methanol, then passed through the Carbopack column. After three washings, the estriol and its conjugates are desorbed and treated as for urine. Mean analytical recoveries of the analytes in any of these body fluids were within about 92-98%, except for estriol-3-sulfate-16 alpha-glucuronide in serum (mean recovery 88.3%). The limit of sensitivity is well below the concentrations of clinical interest, and the method is not susceptible to substantial interferences.

Improved determination of estriol-16 alpha-glucuronide in pregnancy urine by direct liquid chromatography with fluorescence detection.

In this relatively simple, rapid assay of estriol-16 alpha-glucuronide in pregnancy urine, the urine sample is diluted 20-fold with phosphate buffer (pH 5.2) containing 360 mL of acetonitrile and 2 g of cetyltrimethylammonium bromide per liter, then directly injected into the chromatograph. A sample can be assayed within 14 min. Day-to-day CVs ranged from 2.3% at 45 mg/L to 2.9% at 4.8 mg/L. The limit of sensitivity is 0.4 mg/L. Results by the present method (y) correlated and compared very well with those by a method involving fractionation of estrogen conjugates and gas chromatography (x) for 24 samples of pregnancy urine (y = 1.09x + 0.303; r = 0.947). This assay is inexpensive and suitable for complete automation.

Improved assay of unconjugated estriol in maternal serum or plasma by adsorption and liquid chromatography with fluorimetric detection.

In this improved assay only a 500-microL sample is needed and a single assay can be done within 30 min, 10 samples within 90 minutes. The sample of serum or plasma is diluted 20-fold with water, and the estriol is adsorbed from it onto graphitized carbon black ( Carbopack B, Supelco ). After two washings the estriol is desorbed with chloroform/methanol (60/40 by vol), which then is evaporated. The residue is redissolved in 50 microL of water/acetonitrile, and 20 microL is injected into the chromatograph. Analytical recovery for estriol-supplemented serum or plasma averaged 98.6%. Day-to-day CVs ranged from 3.9% at 2 micrograms/L to 2.1% at 20 micrograms/L. The limit of sensitivity is 0.3 micrograms/L, which makes this procedure suitable for determination of estriol even in the first half of pregnancy. Our method is inexpensive, and shows that liquid chromatography can be used to determine estriol in pregnancy serum or plasma. It also is more sensitive and precise and requires less sample than other such methods.

Preliminary isolation of urinary placental estriol before gas or liquid chromatography.

We describe a rapid, simple assay for placental estriol in urine, involving a small sample volume (250 microL) and, correspondingly, a small amount of enzyme reagent. After hydrolysis, estriol is adsorbed from urine onto graphitized carbon black (Carbopack B). After some washing steps, estriol is desorbed with a small volume of a suitable mobile phase. This single-step purification technique is rapid (about 15 min), with minimal sample manipulation. Analytical recoveries for estriol-supplemented urine ranged from 95.9 to 101.0%. Ten replicate analyses of urines containing typical concentrations of estriol gave CVs of 3.3, 2.6, and 2.4% for low, medium, and high concentrations, respectively. The specificity of our extraction technique was good, as assessed by treating 35 urine samples and quantifying estriol by gas chromatography with packed and capillary columns and by liquid chromatography with ultraviolet and fluorometric detection.