Osteoporosis represents an important cause of morbidity in adult rheumatoid arthritis (RA) patients who exhibit increased fracture risk. It is thought that osteoclast and its dysfunction which mediated by many cytokines are the principal pathogenesis of this bone disease, although the mechanisms are still not fully understood. Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL) have been revealed in the pathogenesis of primary osteoporosis and other metabolic bone diseases. Thus, the aim of this study was to investigate the possible role of the OPG/RANKL system in RA-related bone loss. A total of 64 Chinese patients with RA and 60 healthy control subjects were involved. Serum levels of OPG and RANKL were measured by ELISA. BMD of nondominant forearm, lumbar spine(L(1-4)) and proximal femur, including femoral neck, Wards triangle, greater trochanter were assessed using dual-energy X-ray absorptiometry. RA patients had a higher incidence of osteoporosis (23/64, 35.9%) than that in healthy controls (9/60, 15.0%) (P < 0.0001). They displayed lower BMD values than controls at positions of all detected region. Compared with healthy controls, RA group showed significantly higher serum levels of RANKL (48.4 ± 12.5 vs. 23.0 ± 11.2 pmol/l, P < 0.0001), lower serum levels of OPG (106.2 ± 40.6 vs. 231.6 ± 65.6 pg/ml, P < 0.0001), and OPG/RANKL ratio (2.4 ± 0.7 vs. 7.0 ± 1.1, P < 0.0001). Multiple linear regression analysis revealed that in RA group, plasma rheumatoid factor concentration (β = -0.187, P = 0.031), swollen joint count (β = 0.567, P = 0.029), BMD at forearm (β = 0.324, P = 0.002), femoral Wards triangle (β = 0.370, P < 0.0001), and lumbar spine (β = 0.313, P = 0.003) were the contributors for serum OPG (R ( 2 ) = 0.718, P < 0.0001). Age (β = 0.241, P = 0.042) and BMD at femoral Wards triangle (β = -0.441, P < 0.0001) and lumbar spine (β = -0.320, P = 0.013) were the determinants for serum RANKL (R ( 2 ) = 0.616, P < 0.0001), while swollen joint count (β = 1.029, P = 0.019) and BMD at femoral neck (β = 0.285, P = 0.042) for serum OPG/RANKL ratio (R ( 2 ) = 0.279, P < 0.011). Analysis of logistic regression showed age (P = 0.004, OR = 1.156, 95% CI: 1.047-1.276) and the level of C-reactive protein (P = 0.028, OR = 1.019, CI 95%: 1.002-1.036) in peripheral blood of RA were the risk factors for the occurrence of osteoporosis in RA, while OPG/RANKL ratio (P = 0.007, OR = 0.035, CI 95%: 0.003-0.400) was the unique protective factor. These data suggest that, in Chinese RA patients, an altered modulation of the OPG/RANKL system resulting in increased RANKL and decreased OPG in peripheral blood, could contribute to the bone loss characteristic and the generation of osteoporosis in these patients. Changes of ratio of OPG/RANKL might be a protective mechanism against the accelerated bone loss in RA.

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOFMS) for the first time. Liquid-liquid extraction was applied to processing urine samples, and dilute-shoot analyses of intact metabolites were also presented. In LC-QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M-H](-) as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography-mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α-methyl-5α-androst-3-one-17β-sulfate) together with S2 (1α-methyl-5α-androst-17-one-3β-sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse.

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.

In this study, clostebol metabolic profiles were investigated carefully. Clostebol was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (MS) using full scan and targeted MS/MS techniques with accurate mass measurement for the first time. Liquid-liquid extraction and direct injection were applied to processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M-H](-) as target ion in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Fourteen metabolites were found for clostebol, and nine unreported metabolites (two free ones and seven sulfate conjugates) were identified by MS, and their potential structures were proposed based on fragmentation and metabolism pathways. Four glucuronide conjugates were also first reported. All the metabolites were evaluated in terms of how long they could be detected and S1 (4ξ-chloro-5ξ-androst-3ξ-ol-17-one-3ξ-sulfate) was considered to be the long-term metabolite for clostebol misuse detected up to 25 days by liquid-liquid extraction and 14 days by direct injection analysis after oral administration. Five conjugated metabolites (M2, M5, S2, S6 and S7) could also be the alternative biomarkers for clostebol misuse.

In this study, the metabolic profiles of a new illicit drug AMB-FUBINACA were investigated using both human liver microsome and zebrafish models. Liquid chromatography Q Extractive HF Hybrid Quadrupole-Orbitrap mass spectrometry (LC-QE-HF-MS) was employed to analyze the metabolic sites and pathways. AMB-FUBINACA was added to the in vitro liver microsome incubation model to simulate the metabolic processes in human body. The results showed that a total of 17 metabolites were generated in the human liver microsome model; the main metabolic pathways of the phase I metabolism included ester hydrolysis, methylation, ester hydrolysis combined with decarboxylation, hydroxylation, ester hydrolysis combined with indazole ring hydroxylation, etc. while glucuronidation served as the main metabolic pathway of the phase II metabolism. The zebrafish system produced a similar result with 16 of the same 17 metabolites identified. The phase I metabolites M3.1 (ester hydrolysis), M1.2 (alkyl chain hydrolysis) and the phase II metabolite M3.2 (M3.1 glucuronide) were recommended to be the potential poisoning markers.