Adrenal-mediated endogenous metabolites inhibit puberty in female mice.

While assessing a potential role of adrenal glands in the production of the hitherto unidentified puberty-delaying pheromone of female mice, the urinary volatile profiles of normal and adrenalectomized animals were quantitatively compared. Six components, whose concentrations were depressed after adrenalectomy, were identified: 2-heptanone, trans-5-hepten-2-one, trans-4-hepten-2-one, n-pentyl acetate, cis-2-penten-1-yl acetate, and 2,5-dimethylpyrazine. When these laboratory-synthesized chemicals were added (in their natural concentrations) to either previously inactive urine from adrenalectomized females or plain water, the biological activity was fully restored.

Inhibition of glycolytic enzymes by endogenous aldehydes: a possible relation to diabetic neuropathies.

Endogenous saturated and unsaturated aldehydes were found in significant elevations in serum of diabetic humans and rats. These compounds, originating from the lipid peroxidation processes, are shown here to be potent inhibitors of the glycolytic enzymes, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. The inhibition process is non-competitive and progressive. The aldehyde mixture, when supplemented to the standard rat diet at 1/100 ratio, caused nerve damage that is reminiscent of diabetic polyneuropathies.

A unique urinary constituent, 6-hydroxy-6-methyl-3-heptanone, is a pheromone that accelerates puberty in female mice.

BACKGROUND: Olfactorily mediated puberty acceleration in female mice (measured by an increase in uterine weight) has been observed since the 1960s without the active chemosignal being structurally identified. There are many controversies in the literature as to whether this male-originated pheromone is a volatile substance. We investigated the chemical nature of the urinary fractions that are responsible for the characteristic uterine weight increases. RESULTS: The active pheromone was identified as 5,5-dimethyl-2-ethyltetrahydrofuran-2-ol and/or its open-chain tautomer (6-hydroxy-6-methyl-3-heptanone). A series of cyclic vinyl ethers were isolated from chromatographically active fractions of the urine. Because these compounds did not accelerate puberty, we postulated that these ethers were degradation products of a lactol (5,5-dimethyl-2-ethyltetrahydrofuran-2-ol). The lactol was then detected directly in the mouse urine extract using a silylation agent. Synthetic 6-hydroxy-6-methyl-3-heptanone had strong biological activity, whereas its close structural analogs did not. CONCLUSIONS: The male house mouse excretes into its urine a large quantity of a volatile substance that has a unique lactol/hydroxyketone structure. This substance is capable of binding to the less volatile urinary constituents, such as proteins or peptides, and is active in puberty-acceleration bioassays. The controversies regarding the volatility of the puberty-accelerating pheromones can now be explained by considering a complex of volatile lactol/hydroxyketone and urinary proteins.

Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein.

Five structurally diverse small ligands, all binding to the major urinary protein (MUP) of the male house mouse, show individually puberty-accelerating pheromonal activity in the recipient females. A recombinant MUP (identical structurally to the natural protein) has shown no biological activity. While four of these ligands were previously implicated in oestrus synchronization (Whitten effect), the same chemosignals now appear responsible for both sexual maturation and cycling in adult females.

Tandem mass spectrometry of model peptides modified with trans-2-hexenal, a product of lipid peroxidation.

Small molecules formed during lipid peroxidation can react with the basic groups in proteins through different mechanisms. Recently, substituted pyridinium moieties were observed during in vitro incubations of lysine-containing peptides with 2-alkenals. To explore the dissociation behavior of peptides with pyridinium-derivatized lysine residues, the peptide ions created through either matrix-assisted laser desorption/ionization or electrospray ionization were studied with tandem mass spectrometry. The permanently charged pyridinium ions fragment primarily through the charge-remote processes. Under high energy collision-induced dissociation, a number of diagnostic ions were observed that could potentially be used to identify modified residues in proteins. The origins of these ions were studied using deuterium exchange and higher-order mass spectrometry experiments using an ion trap instrument. Rational structures for these ions are proposed.

Quantitative alterations of steroid urinary profiles associated with diabetes mellitus.

Capillary gas chromatography was used for the acquisition of quantitative metabolic profiles of urinary steroids from normal subjects and diabetic patients. Structures of 70 steroid metabolites have been assigned tentatively through mass spectrometry. Quantitative differences between the groups of normal and pathological samples (both males and females) are seen primarily for androsterone, etiocholanolone, dehydroepiandrosterone, 11-oxygenated 17-ketosteroids, corticosteroid metabolites, and a newly discovered metabolite, recently identified as a C26 pentol. The only significant difference between two different age groups of normal males was shown by androsterone.

Urinary profiles of volatile and acid metabolites in germfree and conventional rats.

Qualitative and quantitative differences in the urinary excretion of volatile and acidic metabolites in germfree and conventional rats were examined by capillary gas chromatography and gas chromatography/mass spectrometry. A number of carbonyl compounds, including several short-chain aliphatic ketones and acetophenone, were higher in the conventional urines, while many heterocyclic compounds (furan derivatives, benzothiazole and others) were lower. Both qualitative and quantitative differences were observed in the urinary excretion of acidic metabolites. Three meta-hydroxy phenolic acids appeared only in the conventional rat urines, while levels of many other aromatic and aliphatic acids were also higher.

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.

3-benzoyl-2-quinolinecarboxaldehyde: A novel fluorogenic reagent for the high-sensitivity chromatographic analysis of primary amines.

3-Benzoyl-2-quinolinecarboxaldehyde has been synthesized and characterized for use as a precolumn fluorogenic reagent for the ultrahigh sensitivity determination of primary amines by micro-column liquid chromatography with laser-induced fluorescence detection. The reaction conditions and the spectral properties of the derivatives were investigated with standard amino-acids. The detection limits, with an HeCd laser operated at 442 nm, are in the low femtogram range. The linear dynamic range is at least three orders of magnitude. The separation of a standard amino-acid mixture and the high-sensitivity analysis of a hydrolysed protein sample are demonstrated.

Social housing influences the composition of volatile compounds in the preputial glands of male rats.

In rodents the preputial glands are one of the major sources of pheromones. These volatile chemosignaling compounds are known to elicit specific behavioral and physiological effects in their conspecifics. While social stress can alter both the behavior and hormonal status of rodents, little is known about its influence on the volatile constituents of the preputial glands. We have examined the composition of volatile compounds in the preputial glands of gonadally intact male rats housed for 70 days in either unisex triads (three/cage) or singly. The rank status of triad-housed rats was based on quantitative behavioral assessments taken during the initial 30 min of triad housing. Dominant rats had heavier preputial glands compared to subdominant and subordinate rats. Capillary gas chromatography-mass spectrometry identified 56 volatile preputial compounds, of these 17 did not differ between groups while 26 compounds were significantly higher in the single-housed compared to the triad-housed rats. Six additional volatile compounds were higher in the dominant compared to the other 3 groups, while another six compounds were higher in both the dominant and single-housed rats compared to the subdominant and subordinate rats. It can be concluded that both housing condition and social rank status have significant but different effects on the composition of volatile compounds found in preputial glands of male rats. The physiological and behavioral significance of these changes in preputial gland volatile compound composition in rats remain to be investigated.

Putative chemosignals of the ferret (Mustela furo) associated with individual and gender recognition.

Quantitative stir bar sorptive extraction methods, both in the aqueous and headspace modes, followed by thermal desorption gas chromatography-mass spectrometry were used to investigate individual variations in the volatile components of male and female ferret (Mustela furo) urine. The urinary profiles were further compared with volatile profiles of anal gland secretions of breeding male and female ferrets. Thirty volatile compounds were quantified in male and female urine. Among them, 2-methylquinoline was unique to male urine. Four ketones (4-heptanone, 2-heptanone, o-aminoacetophenone, and a dimethoxyacetophenone) and several nitrogen compounds (e.g., 2,5-dimethylpyrazine, quinoline, 4-methylquinazoline) and low levels of three unidentified nonsulfur compounds were significantly more abundant in males than in females. Quantitative comparison of 30 volatile urinary compounds showed several statistically significant differences between the sexes and individuals of the same sex. These findings suggest that ferrets may use urine marking for sex and individual recognitions. Ten of the 26 compounds identified in anal gland secretions from females and males were also found in urine. However, most of the major compounds (thietanes, dithiolanes, and indole) in anal glands were not present in urine. This suggests that urine may convey specific signals that differ from those of anal glands. Additionally, 10 volatiles (two aldehydes, five ketones, benzothiazole, 2-methylquinoline, and 4-methylquinazoline), not previously identified, were found in ferret anal gland secretions. Among the new compounds, o-aminoacetophenone was found only in males, while only traces of this compound were found in females. Similar results were previously obtained in anal glands of three other Mustela species. These findings provide new information about the constituents of urine and volatile components of anal gland secretions in ferrets.

Formation of cyclic enol ethers from a labile biological precursor: an example of analytical artifacts.

Three isomeric enol ethers are among those constituents apparently unique to mouse urine as identified by gas chromatographic analysis. These compounds appear to be artifacts arising from the cyclization and dehydration of 6-hydroxy-6-methyl-3-heptanone. Identification of the trimethylsilyl ether of 6-hydroxy-6-methyl-3-heptanone in the silylated ether extract of mouse urine indicates that the precursor keto alcohol is indeed present in the urine. Since similar heterocyclic compounds are often identified in urine samples analyzed by gas chromatography, formation of various analysis artifacts arising from analogous cyclization and dehydration reactions is likely.

Effects of starvation and refeeding on the excretion of urinary steroid metabolites in mice with different genetic background.

Gas chromatographic steroid metabolic profiling procedures have been applied to investigations of the effects of starvation and refeeding in mice. Urinary steroid metabolites were quantitatively followed during the starvation-refeeding experiments for mice with different genetic backgrounds. Some quantitative alterations were noted for certain congenic strains of mice. The metabolites which exhibited such quantitative variations were tentatively identified by means of combined gas chromatography-mass spectrometry.

Quantitative alterations in the metabolism of carbonyl compounds due to diet-induced lipid peroxidation in rats.

Following the dietary induction of lipid peroxidation in rats (verified by the levels of malonaldehyde and glutathione peroxidase), the urinary carbonyl compounds were followed chromatographically. Through a headspace gas chromatographic procedure, increases of several aldehydes and furan derivatives were noticed. Liquid chromatography of the dinitrophenylhydrazone derivatives of urinary carbonyls provided a more definitive experiment, in which the increased-peroxidation animals could be compared to those maintained on a control diet. Several carbonyl metabolites, identified by mass spectrometry, were elevated following the lipid peroxidation induction.

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.

Substituted 2-hydroxy-1,2-dihydropyrrol-3-ones: fluorescent markers pertaining to oxidative stress and aging.

Previous observations that the aging process correlates with occurrence of certain fluorescent biological pigments have led to numerous efforts in elucidating the chemical nature of the fluorophores generated through reactions of primary amines and various products of lipid peroxidation. In this study, model reactions of saturated aldehydes with aliphatic amines in the presence of peroxides were found to generate structurally unusual fluorescent compounds. Substitution of a lysine-containing peptide for simpler amines has also yielded similar fluorescence. The spectral excitation and emission maxima (around 360 and 430 nm, respectively) of these fluorophores match those widely reported in peroxidized biological objects. The fluorescent compounds in our model studies have been chromatographically isolated and their structures determined through mass spectrometry, NMR spectrometry, and Fourier-transform infrared spectroscopy. The spectrometric data indicate the fluorescent products to be alkylated 2-hydroxy-1,2-dihydropyrrol-3-ones, obtained by the action of 1,2,4-triketone intermediates upon the primary amines. Independent syntheses of several 1,2,4-triketones were carried out. One such triketone reacted with hexylamine to form a fluorescent compound spectroscopically identical to the fluorescent reaction product of hexanal, hydrogen peroxide, and hexylamine.

Shielding of piezoelectric ultrasonic probes in Hall effect imaging.

This paper addresses significant sources of electromagnetic noise in Hall effect imaging. Hall effect imaging employs large electrical pulses for signal generation and high sensitivity ultrasonic probes for signal reception. Coherent noise arises through various coupling mechanisms between the excitation pulse and the probe. In this paper, the coupling mechanisms are experimentally isolated and theoretically analyzed. Several methods of shielding the probe from electromagnetic interference are devised and tested. These methods are able to reduce the noise to levels below the random thermal noise, thereby improving the signal-to-noise ratio in HEI by two orders of magnitude.

Volatile compounds associated with estrus in mouse urine: potential pheromones.

Female mice that had been made estrous through hormone implantation excreted in their urine significantly enhanced levels of n-pentyl acetate, cis-2-penten-1-yl acetate, p-toluidine, 2-heptanone, and 3 unsaturated ketones. The relationship of these volatiles to a signaling function of the estrous urine is postulated. Structural elucidations of these compounds were carried out through capillary gas chromatography/mass spectrometry and the synthesis of authentic samples.

Ultrasensitive fluorometric detection of carbohydrates as derivatives in mixtures separated by capillary electrophoresis.

Reducing monosaccharides and oligosaccharides, after reductive amination, were separated and detected as their 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) derivatives by capillary electrophoresis/laser-induced fluorescence. Under optimized conditions, the minimum detectable quantities for monosaccharide solutes were assessed at low attomole levels (0.5 amol for the CBQCA derivative of galactose). The system has shown considerable promise for high-sensitivity analysis of both neutral and amino sugars in glycoproteins. Complex oligosaccharides, isolated from bovine fetuin by hydrazinolysis, were also successfully "mapped."

Putative chemical signals from white-tailed deer (Odocoileus virginianus). Urinary and vaginal mucus volatiles excreted by females during breeding season.

Urine and vaginal mucus samples from female white-tailed deer in estrus and mid-cycle were analyzed by combined gas chromatography-mass spectrometry (GC-MS). Forty-four volatiles were found in mucus and 63 in urine. The volatiles common to both vaginal mucus and urine included alcohols, aldehydes, furans, ketones, alkanes, and alkenes. Aromatic hydrocarbons were present only in the vaginal mucus, whereas pyrans, amines, esters, and phenols were found only in urine. Both estrous mucus and estrous urine could be identified by the presence of specific compounds not present in mid-cycle samples. Numerous compounds exhibited dependency on ovarian hormones.