A liquid chromatography/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of 12 tetrahydrocorticosteroid glucuronides in human urine has been developed. The analytes were 3- and 21-monoglucuronides of tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, and their 5alpha-stereoisomers. The mass spectrometric behaviors of these glucuronides in negative-ion ESI-MS/MS revealed the production of intense, structure-specific product ions within the same group of glucuronides. Regioisomeric glucuronides could be distinguished by collision-induced dissociation and tandem mass spectrometry. Using a linear ion trap instrument operating in the negative-ion mode and by monitoring the transition ions of [M - H](-) --> [M - H - CH(2)O](-) for 3-monoglucuronides and [M - H](-) --> [M - H - CH(2)OG](-) for 21-monoglucuronides, a sensitive and specific assay was developed. Initial steps in the assay were a simple solid-phase extraction and the addition of [9,12,12,21,21-d(5)]-tetrahydrocortisone-3-glucuronide (prepared by enzyme-assisted synthesis) as an internal standard. The method was applied to determine the 12 tetrahydrocoticosteroid glucuronides in urine from healthy subjects and from patients with excessive cortisol production. The method described here appears to be useful for clinical and biochemical studies.

As the principal glucocorticoid in humans, cortisol plays a crucial role in modulating the metabolic and homeostatic process that protects against stress, shock, inflammation, etc.3-7) It is synthesized primarily in the zona fasciculata with a small contribution from the zona reticularis.3) While cortisol is essentially secreted by the adrenal glands, cortisone is mainly produced using 11b-hydroxysteroid dehydrogenase isoenzymes, which interconvert cortisol to hormonally inactive cortisone.3) Cortisol and cortisone are extensively metabolized to tetrahydro-reduced derivatives: tetrahydrocortisol (THF) and tetrahydrocortisone (THE), and their 5a-stereoisomer (allo-THF and allo-THE, respectively). 3,[7][8][9] Metabolism decreases the biological activity of hormones and increases their water solubility by converting them to hydrophilic compounds that can be excreted in urine. Unmetabolized cortisol and cortisone comprise only ca. 0.1% of the total urinary cortisol metabolites. At least 90% of the tetrahydro-derivatives of cortisol and cortisone metabolites are excreted into the urine as sulfate or glucuronide conjugates.10-13) Tetrahydro-11-deoxycortisol (THS) and its 5a-stereoisomer (allo-THS) which are tetrahydro-reduced metabolites of 11-deoxycortisol (11-DOC), a biosynthetic precursor of cortisol, are also excreted in urine as conjugated forms. 8,10,12) In order to obtain information regarding the related biochemistry, physiology, and pathophysiology of endocrine disorders, it is necessary to identify all the corticosteroid metabolites found in urine, and determine the exact structure of their conjugates, their concentration, and the dynamics of their formation and disposal.At present, analytical methods for the detection of conjugated tetrahydrocorticosteroids are based on gas chromatography and mass spectrometric determination of hydrolyzed and derivatized compounds.14-18) Although these methods are robust and sensitive, sample preparation in these indirect methods is time consuming, and the GC-MS sample throughput is relatively low. Thus the development or more straightforward methods based on the direct analysis of steroid conjugates without the need for a deconjugation process is of great interest. The application of liquid chromatographic separation interfaced by soft ionization techniques, such as electrospray ionization (ESI) with tandem mass spectrometry and/or ion trap mass spectrometry, offers an effective analytical tool for the direct monitoring of the urinary conjugates of tetrahydrocorticosteroids. 19,20) Recently, we reported a highly sensitive and specific liquid chromatography/ESI-linear ion trap mass spectrometry method for the direct measurement of the glucuronide conjugates of THF, THE, THS, and their 5a-stereoisomer in human urine.21) The authentic glucuronides had been chemically synthesized by Hosoda et al. [22][23][24][25] One significant drawback of sulfate conjugate analysis, however, is still the lack of reference materials, which are essential for the development and application of ...

Reactive metabolic-modified proteins have been proposed to play an important role in the mechanism(s) of the hepatotoxicity and colon cancer of lithocholic acid (LCA). To identify cellular proteins chemically modified with LCA, we have generated a monoclonal antibody that recognizes the 3a-hydroxy-5b-steroid moiety of LCA. The spleen cells from a BALB/c mouse, which was immunized with an immunogen in which the side chain of LCA was coupled to bovine serum albumin (BSA) via a succinic acid spacer, was fused with SP2/0 myeloma cells to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (g2b, k) was specific to LCA-N a -BOC-lysine as well as the amidated and nonamidated forms of LCA. The immunoblot enabled the detection of LCA residues anchored on BSA and lysozyme. The antibody will be useful for monitoring the generation, localization, and capture of proteins tagged with LCA, which may be the cause of LCA-induced toxicity.

Regular ArticlePrion protein (PrP) is a cell-surface glycoprotein implicated in the pathogenesis of a range of neurodegenerative disorders collectively termed transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cows, and chronic wasting disease (CWD) in deer.1-4) PrP exists in two distinct forms: cellular PrP (PrP C ) and a pathogenic or scrapie form (PrP Sc ) derived from PrP C . Although there is no difference in the primary structure of these isoforms, spectroscopic studies revealed that PrP C has a high ahelical content, whereas PrP Sc is composed primarily of bsheets. 5,6) There are many hypotheses regarding the conversion of PrP C to PrP Sc , but the data suggest that conversion is entirely conformational and involves no amino acid substitutions or deletions, and thus supports the protein-only hypothesis.2) Prion replication requires the conversion of PrP C into PrP Sc , where PrP Sc acts as a template and protein X functions as a chaperone. [7][8][9] Mature human PrP (hPrP) consists of 253 amino acids, with a C-terminal glycosylphosphatidylinositol (GPI) anchor and two glycosylation sites (Fig. 1). The N-terminal domain, which includes four repeats of the PHGGGWGQ octapeptide, is a flexibly disordered region. In contrast, the C-terminal domain, which includes two a-helices and a GPI anchor, is a folded region. The middle domain includes two b-sheets and one a-helix.10-12) The hPrP region spanning amino acid residues 106-126 in the middle domain is thought to be responsible for the pathogenic properties of PrP Sc , including neurotoxicity, protease-resistance, induction of hypertrophy, and promotion of astrocyte proliferation. [13][14][15][16][17] Although PrP metal-binding sites have been investigated using full-length PrP C or synthetic fragment peptides and it is now generally accepted that PrP C binds copper in vivo, 18) most researchers have focused on the octarepeat region, [19][20][21][22][23] and there are few reports describing the metal-binding ability of the middle-and C-terminal domains of hPrP.24) The interaction of full-length and truncated forms of PrP with Cu 2+ has been investigated using a range of techniques, including electron paramagnetic resonance (EPR), [25][26][27] We developed a column switch (CS)-HPLC system that can detect direct metal-binding to the octarepeat region of hPrP C , and the data obtained using our CS-HPLC method agreed well with previous CD analyses. 42,43) In this study, we used CS-HPLC to analyze the metal-binding characteristics of 21 synthetic fragment peptides derived from the sequence of hPrP amino acids 60-230. Key words prion protein; metal-binding; metal chelate affinity column; column switch HPLC; synthetic peptide Chem. Pharm. Bull. 59(8) 965-971 (2011)

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