Identification of metabolites of [1,2,3-(13)C]Propargyl alcohol in rat urine by (13)C NMR and mass spectrometry.

Little is known about the metabolism of acetylenic (C&tbd1;C) compounds commonly used in the formulation of pesticides. To better understand the in vivo reactivity of this bond, we examined the metabolism of propargyl alcohol (PA), 2-propyn-1-ol, used extensively in the chemical industry. [1,2,3-(13)C, 2,3-(14)C]PA was administered orally to male Sprague-Dawley rats. Approximately 56% of the dose was excreted in urine by 96 h. Major metabolites were characterized, directly, in the whole urine by one- and two-dimensional (13)C NMR. To determine the complete structures of metabolites of PA, rat urine was also subjected to TLC followed by purification of separated TLC bands on HPLC. The purified metabolites were identified by (13)C NMR and mass spectrometry and by comparison with available synthetic standards. The proposed metabolic pathway involves oxidation of propargyl alcohol to 2-propynoic acid and further detoxification via glutathione conjugation to yield as final products: 3, 3-bis[(2-(acetylamino)-2-carboxyethyl)thio]-1-propanol, 3-(carboxymethylthio)-2-propenoic acid, 3-(methylsulfinyl)-2-(methylthio)-2-propenoic acid, 3-[[2-(acetylamino)-2-carboxyethyl]thio]-3-[(2-amino-2-carboxyethyl)t hio]-1-propanol and 3-[[2-(acetylamino)-2-carboxyethyl]sulfinyl]-3-[2-(acetylamino)-2-car boxyethyl]thio]-1-propanol. These unique metabolites have not been reported previously and represent the first example of multiple glutathione additions to the carbon-carbon triple bond.

Hyperalgesia to heat after intradermal injection of capsaicin.

Capsaicin injected intradermally into the human forearm lowered the pain threshold for heat at the injection site. Both the magnitude and duration of hyperalgesia were dose dependent over the range of 0.1-100 micrograms, given in a constant volume of 10 microliter. Thus, capsaicin may be a useful tool in studies of the neural mechanisms of hyperalgesia.

The magnitude and duration of itch produced by intracutaneous injections of histamine.

The magnitude and duration of itch sensation produced by intracutaneous injection of histamine were determined for humans with the procedure of magnitude estimation scaling. Thirteen subjects received a 10-microliter intracutaneous injection of histamine at doses of 0.0001, 0.001, 0.01, 0.1, 1, and 10 micrograms into the volar forearm; eight of these subjects also received a 100-microgram dose. One subject received multiple injections over several weeks to determine the reliability of the magnitude estimates of itch. Following each injection, the area of flare and duration of itch were also determined. Intracutaneous injection of histamine produced a pure sensation of itch, without pain. The magnitude of itch increased in a dose-dependent fashion. The lowest histamine dose that produced itch greater than the itch produced by vehicle was 0.01 micrograms. The greatest itch was produced by the 100-microgram dose. A power function fitted to the mean magnitude estimates had an exponent of 0.17, indicating a negatively accelerating relation between the magnitude of itch and histamine dose. The one subject who received histamine over several weeks gave fairly reproducible estimates of itch magnitude. The duration of itch and the area of flare also increased in a dose-dependent fashion. The lowest dose of histamine that produced a duration of itch longer than the itch produced by the vehicle was 0.1 microgram, while the 100-microgram dose produced the longest duration of itch. Although the area of flare increased with each increase in dose from 0.1 to 10 micrograms, the areas of flare produced by 10 and 100 micrograms of histamine did not differ. These results indicate that humans can scale the magnitude of itch produced by histamine in a dose-dependent manner. In addition, the duration of itch and the area of flare produced by histamine are dose-dependent, confirming results of previous investigators. Intracutaneous histamine is easily quantifiable and may thus be a useful stimulus in neurophysiological studies of the peripheral neural mechanisms of itch.

Intravitreal injection of purified human chorionic gonadotropin lowers IOP in rabbits.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits alpha and beta held together by noncovalent forces. Each subunit contains about 30% carbohydrate and is extensively crosslinked by disulfide bonds. Previous work from our laboratory with commercial preparations of hCG indicated that intravitreal injection of hCG lowered intraocular pressure (IOP). Our work has been extended by using purified hCG obtained by reverse phase high performance liquid chromatography of a commercial preparation. With a wide pore octyl silica column and a step gradient composed of dilute aqueous trifluoroacetic acid and methanol, several peaks were obtained. The major peak was shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis and amino acid analysis to contain both alpha and beta subunits. That this major peak contained intact hormone rather than a mixture of subunits was revealed by its ability to enhance the fluorescence of 8-anilino-1-naphthalene sulfonate and stimulate the release of cyclic AMP from isolated rat testes; subunits of hCG lack these properties. Physiological doses of hCG from this major peak injected intravitreally in rabbit eyes resulted in significant decreases in IOP without associated irritation when compared with contralateral control eyes.

Lack of metabolism of timolol by ocular tissues.

The ocular metabolism of timolol has been studied in the iris/ciliary body, vitreous humor, neuro-retina and cornea from an albino rabbit, pigmented rabbit and cynomolgus monkey by incubating tissues isolated from these animals with radiolabeled drug for 20 hours at 37 degrees C in Hanks' balanced salts solution. After removal of the tissues by centrifugation and subsequent deproteinization, supernatants were mixed with unlabeled timolol and analyzed by isocratic reverse phase high performance liquid chromatography with liquid scintillation counting and ultraviolet detection. Although this analytical system readily demonstrated the production of timolol metabolites by rat liver, the production of timolol metabolites by ocular tissues could not be demonstrated. With most samples high recoveries of unmetabolized timolol were obtained. The lowest level of recovery of unmetabolized drug as compared to a control (drug added after the tissue had been incubated) was obtained for the iris/ciliary body from the pigmented rabbit suggesting that pigment had bound the drug. Our results suggest that deleterious side effects sometimes seen with the drug or its lack of efficacy with rabbits is not due to ocular metabolism by these tissues.

Preparation, isolation, and immunochemical studies of the cyanogen bromide peptides from a retinal photoreceptor cell autoantigen, S-antigen.

Purified bovine retinal S-antigen (50,000 m.w.) was treated with cyanogen bromide, producing seven major and several minor fragments. Six of the major and one of the minor components were isolated by reverse phase high performance liquid chromatography. The peptides were characterized with respect to size by urea-SDS-gel electrophoresis, by amino acid composition, and by their ability to bind antibodies, raised in rabbits immunized with purified bovine S-antigen, in both competition and direct enzyme-linked immunosorbent assays. Four of the purified peptides were found, by the direct assay, to bind antibodies in immune sera raised to the intact antigen. Peptides that were negative, or only weakly bound, in the direct enzyme immunoassay were subsequently conjugated to a carrier, poly-L-Glu-Ala-Tyr, and were retested in the enzyme immunoassay in which a peptide of about 25 residues was also found to contain an antigenic determinant. The same five peptides were positive in the competition assays. Isolation of the peptides and gel electrophoresis under reducing and nonreducing conditions revealed that two of the peptides in the reaction mixture were joined by a disulfide linkage.

In vitro sorption of aluminum complex to guinea pig stratum corneum.

The sorption of aluminum complexes to guinea pig stratum corneum has been studied using our previously described fluorometric and atomic absorption spectrophotometric procedures. The sorption, desorption, and binding properties of the two aluminum systems most often used in topically applied antiperspirants, aluminum chloride and aluminum chlorohydrate, Al2(OH)5Cl . 2H2O were examined as a function of aluminum concentration, sorption time, state of hydration, and for various delipidized tissue specimens. The results indicate rapid uptake of aluminum species in both systems from aqueous solutions for partially hydrated tissue, reaching 50% saturation levels in about 30 min. Pseudo-equilibrium sorption isotherms follow a Langmuir-type sorption behavior over the 10(-4) M to 5 x 10(-3) M aluminum concentration range for both systems reaching plateau sorption capacities. At higher aluminum concentrations, however, the aluminum chlorohydrate isotherm exhibits a long linear increase in sorption following this initial plateau. Sorption of the various aluminum species depends on the hydration state of the tissue with increases in sorption of 2- to 3-fold over tissue prehydration time periods of 0-96 hr. Desorption studies indicate significant reversibility of aluminum chloride sorption from partially hydrated tissue but little desorption from fully hydrated tissue. In contrast, little desorption is observed with aluminum chlorohydrate regardless of tissue hydration levels. These differences are interpreted in terms of the inherent physical-chemical properties of the species contained in these two aqueous aluminum (III) ion systems.

Synthesis and secretion of human chorionic gonadotropin subunits by cultured human malignant cells.

The synthesis and secretion of human chorionic gonadotropin (hCG) subunits have been studied by pulse-chase techniques in JAR choriocarcinoma cells, which produce both the alpha and beta subunits of hCG "eutopically," that is, as part of their expected repertoire of gene products, and in three cell lines that produce either alpha or beta subunit "ectopically." JAR cells contain two intracellular forms of the alpha subunit (Mr = 15,000 and 18,000) and of the beta subunit (Mr = 18,000 and 24,000) but do not accumulate fully processed, "mature" alpha (Mr = 22,000) or beta (Mr = 34,000) subunits during chase of pulse-labeled cells. Mature subunits, however, are secreted into the culture medium during this time. Thus, secretion of mature subunits appears to occur rapidly after processing of the intracellular forms. Two cell lines that ectopically secrete alpha but not beta subunit, ChaGo bronchogenic carcinoma cells and HeLa S3 cervical carcinoma cells, also contain the Mr = 15,000 and 18,000 intracellular forms of alpha subunit and appear to accumulate mature alpha subunit intracellularly. The CBT cell line, derived from a glioblastoma multiforme, produces the Mr = 18,000 and 24,000 intracellular forms of beta subunit with no evidence for alpha subunit synthesis or secretion. These four cell lines should provide "biologic reagents" for the further study of alpha and beta subunit processing and secretion.