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Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free ?-subunit, and ?-subunit core fragment in urine.

ABSTRACT: BACKGROUND:Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free ?-subunit (hCG?), and ?-subunit core fragment (hCG?cf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential immunoextraction method with LC-MS/MS detection for quantification of intact hCG, hCG?, and hCG?cf in urine. METHODS:hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCG? and hCG?cf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. RESULTS:The method was linear to 20 IU/L. Total imprecision was 6.6%-13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4-15.5 IU/L higher than LC-MS/MS results. CONCLUSIONS:The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCG? separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

SUBMITTER: Woldemariam GA

PROVIDER: S-EPMC4334569 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free β-subunit, and β-subunit core fragment in urine.

Woldemariam Getachew A GA Butch Anthony W AW

Clinical chemistry 20140604 8

<h4>Background</h4>Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs amo ...[more]

PMID: 24899693

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Project description:With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.

Project description:During histiotrophic nutrition of the embryo, maternal platelets may be the first circulating maternal cells that find their way into the placental intervillous space through narrow intertrophoblastic gaps within the plugs of spiral arteries. Activation of platelets at the maternal-fetal interface can influence trophoblast behavior and has been implicated in serious pregnancy pathologies. Here, we show that platelet-derived factors impaired expression and secretion of the human chorionic gonadotropin beta-subunit (?hCG) in human first trimester placental explants and the trophoblast cell line BeWo. Impaired ?hCG synthesis was not the consequence of hampered morphological differentiation, as assessed by analysis of differentiation-associated genes and electron microscopy. Platelet-derived factors did not affect intracellular cAMP levels and phosphorylation of CREB, but activated Smad3 and its downstream-target plasminogen activator inhibitor (PAI)-1 in forskolin-induced BeWo cell differentiation. While TGF-? type I receptor inhibitor SB431542 did not restore impaired ?hCG production in response to platelet-derived factors, Smad3 inhibitor SIS3 interfered with CREB activation, suggesting an interaction of cAMP/CREB and Smad3 signaling. Sequestration of transcription co-activators CBP/p300, known to bind both CREB and Smad3, may limit ?hCG production, since CBP/p300 inhibitor C646 significantly restricted its forskolin-induced upregulation. In conclusion, our study suggests that degranulation of maternal platelets at the early maternal-fetal interface can impair placental ?hCG production, without substantially affecting morphological and biochemical differentiation of villous trophoblasts. KEY MESSAGES: Maternal platelets can be detected on the surface of the placental villi and in intercellular gaps of trophoblast cell columns from gestational week 5 onwards. Platelet-derived factors impair hCG synthesis in human first trimester placenta. Platelet-derived factors activate Smad3 in trophoblasts. Smad3 inhibitor SIS3 interferes with forskolin-induced CREB signaling. Sequestration of CBP/p300 by activated Smad3 may limit placental hCG production.

Project description:Immunopathological outcomes in Systemic Lupus Erythematosus (SLE; or lupus) are believed to be autoantibody-mediated. Conditions which promote a Th2 skew (such as pregnancy) should encourage antibody production, worsening antibody-mediated diseases while ameliorating Th1/Th17-mediated diseases. Although an increased propensity toward autoreactivity can be observed in pregnant lupus patients and in pregnant lupus-prone mice, whether a unique human pregnancy-specific factor can contribute to such effects is unknown. This study assessed whether human chorionic gonadotropin (hCG, a pregnancy-specific hormone of diverse function) at physiological concentrations could mediate stimulatory influences on immune parameters in non-pregnant, lupus-prone mice, in light of the hormone's ameliorating effects on Th1-mediated autoimmunity in murine models. Results demonstrate that administration of hCG heightened global autoreactivity in such mice; antibodies to dsDNA, RNP68, Protein S, Protein C, ?2-glycoprotein 1, and several phospholipids were enhanced, and hormone administration had adverse effects on animal survival. Specifically in splenic cell cultures containing cells derived from lupus-prone mice, hCG demonstrated synergistic effects with TLR ligands (up-modulation of costimulatory markers on B cells) as well as with TCR stimuli (enhanced proliferative responses, enhanced levels of cytokines, and the phosphorylation of p38). In both instances, enhanced synthesis of lupus-associated cytokines was observed, in addition to the heightened generation of autoantibodies reactive toward apoptotic blebs. These results suggest that selective transducive, proliferative, and differentiative effects of hCG on adaptive immune cells may drive autoreactive responses in a lupus environment, and may also potentially provide insights into the association between the presence of higher hCG levels (or the administration of hCG) with the presence (or appearance) of humoral autoimmunity.

Dataset Information

Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free ?-subunit, and ?-subunit core fragment in urine.

Publications

Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free β-subunit, and β-subunit core fragment in urine.

Similar Datasets

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