Kouwa Yamashita

Regional Distribution of Prostaglandins D2, E2, and F2α and Related Enzymes in Postmortem Human Brain

Abstract: The presence of prostaglandins D2, E2, and F2α was demonstrated and their contents measured in various regions of postmortem human brain, pineal body, and pituitary by using specific radioimmunoassays and gas chromatography‐mass spectrometry. The three prostaglandins were widely distributed in similar concentrations ranging from several hundred pg/g wet weight to about 40 ng/g wet weight. Prostaglandins D2 and E2 showed consistent and similar regional distributions in all six brains tested; amounts were high in pineal body, pituitary, olfactory bulb, and hypothalamus. On the other hand, prostaglandin F2α was distributed more evenly. Prosta‐ glandin D synthetase and prostaglandin E synthetase activities were found in cerebrum homogenate from a single subject and were recovered from the 100,000 ×g supernatant. The presence of 1 mM glutathione, reduced form, markedly stimulated the activity of prostaglandin E synthetase, but did not affect prostaglandin D synthetase activity. Activity of 15‐hydroxyprostaglandin dehydrogenase was found in the cerebrum homogenate and was partially purified. This enzyme required NADP as a cofactor and copurified with prostaglandin E 9‐ketoreductase.

Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.

Development of highly sensitive quantification method for testosterone and dihydrotestosterone in human serum and prostate tissue by liquid chromatography-electrospray ionization tandem mass spectrometry.

We developed highly sensitive detection of testosterone (T) and 5alpha-dihydrotestosterone (DHT) by liquid chromatography-electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17beta-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN-MeOH-H(2)O-formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.2 ml in serum, and T and DHT with LOQs of 0.5 pg and 1 pg/3mg in prostate tissue, respectively, under acceptable assay performance (intra-assay and inter-assay accuracy and precision). The present method provides reliable and reproducible results for quantification of T and DHT in small volumes of serum and prostate samples for diagnosis in prostatic disorders and male climacteric.

Synthesis of pyridine-carboxylate derivatives of hydroxysteroids for liquid chromatography–electrospray ionization-mass spectrometry

Synthesis and liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) behaviors of the picolinoyl, 6-methylpicolinoyl, nicotinoyl, 2-methoxynicotinoyl and isonicotinoyl derivatives of the hydroxysteroids estrone, estradiol, 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) and testosterone in positive mode were investigated. Each steroid was converted to the corresponding pyridine-carboxylate derivative by the acyl chloride method or the mixed anhydride method using the corresponding free acids and 2-methyl-6-nitrobenzoic anhydride; in each case, the latter method principally gave a better yield. The pyridine-carboxylate derivative of each steroid exhibited a clear single peak in liquid chromatography with a reversed phase column and CH(3)CN-0.1% CH(3)COOH as a mobile phase. The positive-ESI-mass spectra of the picolinoyl, 6-methylpicolinoyl and 2-methoxynicotinoyl derivatives showed a predominance of [M+H](+), whereas [M+H+CH(3)CN](+) was observed with high intensity in the nicotinoyl and isonicotinoyl derivatives. Even in the case of estradiol, with its two hydroxyl groups, a single charged ion of [M+H](+) or [M+H+CH(3)CN](+) was observed in the positive-ESI-mass spectrum of each derivative. The results revealed that picolinoyl derivatization is a simple and versatile method suitable for the sensitive and specific determination of hydroxysteroids by LC-ESI-MS (selected reaction monitoring).

Development of sensitive derivatization method for aldosterone in liquid chromatography–electrospray ionization tandem mass spectrometry of corticosteroids

A highly sensitive quantification method of aldosterone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using recently developed picolinyl derivatization. Aldosterone was smoothly and quantitatively converted to the ethyl ether-picolinyl derivative by treatment with HCl-ethanol followed by the esterification with picolinic acid in the presence of 2-methyl-6-nitrobenzoic anhydride and 4-dimethylaminopyridine. The positive ion-ESI mass spectrum of the ethyl ether-picolinyl derivative was characterized by an appearance of protonated molecule ([M+H](+)) as a base peak. The ethyl ether-picolinyl derivatization gave a successful result in a separation of aldosterone from corticosterone, dehydrocorticosterone and cortexolone, and also provided an approximately 10-fold higher ESI response in the positive-LC-ESI-MS/MS (selected reaction monitoring; SRM) when compared to that of underivatized molecule (negative mode). The limit of quantification of aldosterone by SRM using ethyl ether-picolinyl derivatization (m/z 494-->m/z 448) was 1 pg/0.2 ml serum with accuracy and precision of 92.6% and 5.6%, respectively.

A heterologous enzyme immunoassay of prostaglandin E2 using a stable enzyme-labeled hapten mimic

A sensitive heterologous enzyme immunoassay for prostaglandin E2 was developed using 9-deoxy-9-methylene-prostaglandin F2 alpha as a stable prostaglandin E2 mimic. beta-Galactosidase was conjugated to the hapten mimic. Anti-prostaglandin E2 IgG was bound to a polystyrene tube. The enzyme-labeled hapten mimic mixed with unlabeled prostaglandin E2 was allowed to react in a competitive manner with the immobilized antibody. Then, the beta-galactosidase specifically bound to the antibody was assayed fluorometrically, and the enzyme activity was correlated with the amount of unlabeled prostaglandin E2. According to the calibration curve thus obtained, prostaglandin E2 could be determined in a range of 1.2-430 fmol. Prostaglandin E2 was extracted from human urine by the use of an octadecylsilyl silica column. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high content of prostaglandin E2. The interfering substance was separated from prostaglandin E2 by reverse-phase high-performance liquid chromatography. The purified urinary extract was examined by the enzyme immunoassay for prostaglandin E2, and the validity of the results was confirmed by gas chromatography-selected ion monitoring.

18-Oxocortisol Measurement in Adrenal Vein Sampling as a Biomarker for Subclassifying Primary Aldosteronism

CONTEXT 18-Oxocortisol (18-oxoF) is a derivative of cortisol (F) that is produced by aldosterone synthase (CYP11B2). The potential for this steroid as a biomarker for differentiating patients with aldosterone-producing adenoma (APA) from those with idiopathic hyperaldosteronism (IHA) has not been examined. OBJECTIVES We measured 18-oxoF, aldosterone, and F in plasma from adrenal vein sampling (AVS) of patients with primary aldosteronism. We compared 18-oxoF levels and 18-oxoF/F ratios for their potential to differentiate APA from IHA. DESIGN, SETTING, AND SUBJECTS This study measured 18-oxoF, F, and aldosterone in AVS obtained from patients with unilateral APA (14 cases) or bilateral IHA (seven cases, 14 samples total) using liquid chromatography-tandem mass spectrometry and RIA analyses. RESULTS The levels of 18-oxoF and the ratios of 18-oxoF/F, before and after ACTH stimulation, were significantly higher in blood-draining APA than in those from the contralateral adrenal glands and from adrenal glands with IHA. CONCLUSIONS The 18-oxoF levels and ratios of 18-oxoF/F in AVS samples can be a clinically useful biomarker for differentiating APA from IHA and for determining the localization or lateralization of APA in patients with primary aldosteronism.

Tenuipyrone, a novel skeletal polyketide from the entomopathogenic fungus, Isaria tenuipes, cultivated in the presence of epigenetic modifiers.

The concomitant addition of the histone deacetylase inhibitor and the DNA methyltransferase inhibitor to the culture medium of an entomopathogenic fungus, Isaria tenuipes, greatly enhanced its secondary metabolite production and led to the isolation of tenuipyrone (1), a novel polyketide with an unprecedented tetracyclic ring system bearing a spiroketal structural component, along with two known C(10)-polyketides, cephalosporolide B (2), which is a plausible biosynthetic precursor of 1, and cephalosporolide F (3).

Simultaneous determination of tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

Simultaneous quantification method of three major metabolites of cortisone and cortisol, tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using a recently developed picolinyl derivatization. Conversion of each steroid into the corresponding picolinyl derivatives (1b, 2b or 3b) was performed by mixed anhydride method using picolinic acids and 2-methyl-6-nitrobenzoic anhydride. Derivatization proceeded smoothly to afford the corresponding 3, 21-dipicolinyl derivatives. Positive ion-ESI mass spectra of the picolinyl derivatives were dominated by an appearance of [M+H](+) as base peaks in all cases. The picolinyl derivatives provided 15 to 80-fold higher ESI response in the LC-ESI-MS/MS (selected reaction monitoring: SRM) when compared to those of underivatized molecules in a positive LC-ESI mode. The use of the picolinyl ester, solid-phase extraction, and deuterium labeled internal standards enabled the concentrations of these metabolites in human urine to be determined simultaneously by LC-ESI-MS/MS (SRM) with a small sample volume of less than 1microl urine.

Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is a powerful method for the microanalysis of compounds in biological samples. Compared with gas chromatography-mass spectrometry (GC-MS), this method is more broadly applicable to various compounds and usually does not require a derivatization step before analysis. However, when neutral sterols are analyzed, the sensitivities of usual HPLC-MS/MS method are not superior to those of GC-MS because the sterols are relatively resistant to ionization. In this review, we introduce the recent development of HPLC-MS/MS analysis for the quantification of non-cholesterol sterols. By adding an effective derivatization step to the conventional procedure, sterol analysis by HPLC-MS/MS surpassed that obtained by GC-MS in sensitivity. In addition, sufficient specificity of this method was achieved by selected reaction monitoring (SRM) and thorough chromatographic separation of each sterol.