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5 alpha-Dihydrotestosterone has been widely measured in human prostatic tissue using RIA since it is involved in the pathogenesis of human prostatic hyperplasia and seems to be the best index for the follow-up of patients affected by prostatic cancer under endocrine treatment. A GC-MS method for the simultaneous determination of testosterone (T), 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) in prostatic tissue based on the isotopic dilution technique was developed. Tri-deuterated internal standards of each compound were previously synthetized in our laboratory. After extraction and purification on Sep-Pak C18 and Sephadex LH-20, T and its metabolites were measured as heptafluorobutyric ester (HFB) derivatives. Quantitative analysis was performed on a VG 7070 EQ mass spectromer equipped with a fused silica capillary column using the Selected Ion Monitoring technique. Steroid values (mean +/- SD; ng/g tissue) found in nine human hypertrophic prostates were: T: 0.71 +/- 0.43; DHT: 4.46 +/- 1.41; 3 alpha-diol: 0.34 +/- 0.23. Preliminary results obtained from the detection of the three androgens in human prostatic hyperplasia treated for 3 months with GnRH before surgery seem to indicate that DHT concentration decreases more than 10 times. Values obtained (n = 1; ng/g tissue) were: T: 0.194; DHT: 0.255; 3 alpha-diol: 0.015.

Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of two of the other major adducts were determined by concerted application of 13C-1H two-dimensional chemical-shift correlation, fast-atom-bombardment mass spectrometry and tandem mass spectrometry to the adduct mixtures in aqueous solution.

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.

Horseradish peroxidase and bovine lactoperoxidase (EC 1.11.1.7), when incubated aerobically with arachidonate, gave rise to the formation of substances identified by bioassay as prostaglandin F2 alpha (PGF2 alpha)- and prostaglandin E2 (PGE2)-like compounds. Boiling of enzymes, which suppressed their capacity to peroxidize guaiacol, also destroyed their capacity to convert arachidonate into PG-like compounds. The rates of formation of PG-like compounds rapidly declined with time, approaching zero after 10 and 20 min for PGE2 alpha- and PGE2-like compounds, respectively. Addition of more enzyme further promoted the reaction. Horseradish and lacto-peroxidases showed optimum pH values of 9.0 and 10.0, respectively. Both enzymes exhibited apparent Km values of about 5 x 10(-5) M for arachidonate. Some reducing agents such as ascorbic acid, NADH and adrenaline dose-dependently inhibited this reaction. The haem poison, phenylhydrazine, also inhibited, with an IC50 of 1 x 10(-7) M. Indomethacin inhibited only the formation of PGE2-like compounds with an IC50 of about 3 x 10(-6) M. As compared to a standard commercial preparation of horseradish peroxidase, the purified horseradish basic and acidic isoenzymes exhibited a higher activity, towards arachidonate whereas other haemoproteins, possessing peroxidase activity, were less active. TLC and GC-MS analyses performed on the reaction products led to the identification of PGF2 alpha, PGE2 and PG6K1 alpha and other unidentified arachidonate derivatives. At 25 degrees, pH 9.5, horseradish peroxidase, acting on saturating concentration of arachidonate, catalysed the formation of 60 mumol/min/mmole enzyme of PGE2 + PGF2 alpha. This appears to be the first report of the synthesis of prostaglandins catalysed by peroxidases.

The effect of varying the length of the alkyl bridge linking the chemiluminescent label isoluminol to progesterone on the light yield and binding affinity of progesterone chemiluminescent-marker conjugates was investigated. For this purpose five different derivatives of isoluminol, aminoethyl isoluminol (AEI), aminoethylethyl isoluminol (AEEI), aminobutyl isoluminol (ABI), aminobutyl-ethyl isoluminol (ABEI) and aminoethyl-ethyl isoluminol (AHEI) were covalently linked through a peptide bond to progesterone-11 alpha -hemisuccinate (P-11-HS). The resulting progesterone chemiluminescent-marker conjugates were then evaluated as potential labels for the development of an immunoassay based on monitoring chemiluminescence. These conjugates were able to compete with tritiated progesterone for the binding sites of an antibody raised against progesterone-11 alpha-HS bovine serum albumin, and they showed higher affinity than unaltered progesterone. These conjugates produced light upon oxidation by a hydrogen peroxide-microperoxidase system. The chemiluminescent reaction was optimized in terms of pH, concentration of oxidant, catalyst and conjugate design. Under optimal conditions, all the conjugates were detectable at femtomolar levels. The lowest detection limit was obtained using P-11-HS-ABEI (0.1 fmol). These results indicated that immunoassay techniques based on chemiluminescence can be developed with these labels.

The medicinally important quaternary ammonium salts benzyldimethyltetradecylammonium chloride (BDTA), cetylpyridinium chloride and benzethonium chloride, all afford, under fast atom bombardment (FAB) mass spectrometric conditions, abundant and persistent [M-Cl]+ species usefully amenable to quantitative analysis with the aid of thioglycerol as a liquid FAB matrix. The use of BDTA as an internal standard allowed a direct, precise and accurate determination of cetylpyridinium and benzethonium chlorides, either as pure samples or in dosage forms, in the concentration range 0.05-2 mg/ml.

The oxygenated compounds of Pinus mugo Turra essential oil were investigated by a combination of GC and dry column chromatography (DCC) coordinated by GC data processing. The collected data resulted in a bar graph ("normalized" gas chromatogram) giving the RRT's and relative amounts of 68 components; 38 of them were identified by MS and IR. The described procedure may be used for essential oil analysis in general.

We describe a mass-spectrometric method based on the fast atom bombardment ionization technique in the negative-ion mode for measuring pregnanediol-3 alpha-glucuronide in diluted urine from women. The procedure requires addition of testosterone-17 beta-D-glucuronide (2.5 micrograms/25 microL) to the urine sample as internal standard, and the sample is added directly to the fast atom bombardment target with no further manipulation. We have assessed and evaluated the method by the traditional criteria of reliability.

The release of endogenous aspartic, glutamic, and gamma-aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometric method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+-dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2] from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation-evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.