Characterization and quantitation of a sulfoconjugated metabolite for detection of methyltestosterone misuse and direct identification by LC-MS.

Methyltestosterone (MT) is one of the most frequently misused anabolic androgenic steroids detected in doping control analysis. The metabolism of MT in humans leads to several phase І metabolites and their corresponding phase Ⅱ conjugates. Previous studies have postulated the 3α-sulfoconjugate of 17α-methyl-5ß-androstane-3α,17ß-diol (S2) as principal sulfate metabolite of MT, with a detection window exceeding 10 days. However, a final direct and unambiguous confirmation of the structure of this metabolite is missing until now. In this study, we established an approach to detect and identify S2, using intact analysis by liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) without complex sample pretreatment. An in vitro study yielded the LC-MS/MS reference retention times of all 3-sulfated 17-methylandrostane-3,17-diol diastereomers, allowing for accurate structure assignment of potentially detected metabolites. In an in vivo excretion study with a single healthy male volunteer, the presence of the metabolite S2 was confirmed after a single oral dose of 10 mg MT. The reference standard was chemically synthesized, characterized by accurate mass mass spectrometry (MS) and nuclear magnetic resonance (NMR), and quantified by quantitative NMR (qNMR). Thus, this study finally provides accurate structure information on the S2 metabolite and a direct analytical method for detection of MT misuse. The availability of the reference material is expected to facilitate further evaluation and subsequent analytical method validation in anti-doping research.

Novel dummy molecularly imprinted polymer for simultaneous solid-phase extraction of stanozolol metabolites from urine.

Stanozolol, a synthetic derivative of testosterone, is one of the common doping drugs among athletes and bodybuilders. It is metabolized to a large extent and metabolites are detected in urine for a longer duration than the parent compound. In this study, a novel dummy molecularly imprinted polymer (DMIP) is developed as a sorbent for solid-phase extraction of stanozolol metabolites from spiked human urine samples. The optimized DMIP is composed of stanozolol as the dummy template, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker in a ratio of 1:10:80. The extracted analytes were quantitively determined using a newly developed and validated ultrahigh-performance liquid chromatography tandem mass spectrometry method, where the limits of detection and quantitation were 0.91 and 1.81 ng mL-1, respectively, fulfilling the minimum required performance limit decided on by the World Anti-Doping Agency. The mean percentage extraction recoveries for 3'-hydroxystanozolol, 4ß-hydroxystanozolol, and 16ß-hydroxystanozolol are 97.80% ± 13.80, 83.16% ± 7.50, and 69.98% ± 2.02, respectively. As such, the developed DMISPE can serve as an efficient cost-effective tool for doping and regulatory agencies for simultaneous clean-up of the stanozolol metabolites prior to their quantification.

Anabolic steroids in livestock production: Background and implications for chemical food safety.

The use of steroids in livestock animals is a source of concern for consumers because of the risks associated with the presence of their residues in foodstuffs of animal origin. Technological advances such as mass spectrometry have made it possible to play a fundamental role in controlling such practices, firstly for the discovery of marker metabolites but also for the monitoring of these compounds under the regulatory framework. Current control strategies rely on the monitoring of either the parent drug or its metabolites in various matrices of interest. As some of these steroids also have an endogenous status specific strategies have to be applied for control purposes. This review aims to provide a comprehensive and up-to-date knowledge of analytical strategies, whether targeted or non-targeted, and whether they focus on markers of exposure or effect in the specific context of chemical food safety regarding the use of anabolic steroids in livestock. The role of new approaches in data acquisition (e.g. ion mobility), processing and analysis, (e.g. molecular networking), is also discussed.

Tmem119 is involved in bone anabolic effects of PTH through enhanced osteoblastic bone formation in mice.

The intermittent administration of parathyroid hormone (PTH) exerts potent bone anabolic effects, which increase bone mineral density (BMD) and reduce fracture risk in osteoporotic patients. However, the underlying mechanisms remain unclear. Tmem119 has been proposed as a factor that is closely linked to the osteoblast phenotype, and we previously reported that PTH enhanced the expression of Tmem119 in mouse osteoblastic cells. However, roles of Tmem119 in the bone anabolic effects of PTH in vivo remain unknown. We herein investigated the roles of Tmem119 in bone anabolic effects of PTH using Tmem119-deficient mice. Tmem119 deficiency significantly reduced PTH-induced increases in trabecular bone volume and cortical BMD of femurs. Effects of Tmem119 deficiency on bone mass seemed predominant in female mice. Histomorphometric analyses with calcein labeling showed that Tmem119 deficiency significantly attenuated PTH-induced increases in the rates of bone formation and mineralization as well as numbers of osteoblasts. Moreover, Tmem119 deficiency significantly blunted PTH-induced decreases in phosphorylation of ß-catenin and increases in alkaline phosphatase activity in osteoblasts. In conclusion, the present results indicate that Tmem119 is involved in bone anabolic effects of PTH through osteoblastic bone formation partly related to canonical Wnt-ß-catenin signaling in mice.

Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

In-depth metabolic study of nonsteroidal selective androgen receptor modulator GSK2881078 in thoroughbred horses and horse liver microsomes for doping control.

Nonsteroidal selective androgen receptor modulators (SARMs) are a novel class of compounds that have not yet been clinically approved; however, they appear to have a better anabolic/androgenic ratio than steroids and cause slighter side effects. Sports drug testing laboratories are required to maintain continuously updated doping control analytical methods in light of the widespread misuse of SARMs in elite and amateur sports. This paper describes the metabolic conversion of SARM GSK2881078 in thoroughbred horses following oral administration and in vitro with equine liver microsomes. A liquid chromatography-high-resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. A total of five (M1-M5) in vivo metabolites and six (M1-M6) in vitro metabolites were detected under experimental conditions. Phase I metabolites mainly result from hydroxylation. Methoxylated and side-chain dissociated metabolites were also detected. Neither sulfonic acid nor glucuronic acid conjugated metabolites were observed in this study. Data reported here could aid in the detection of nonsteroidal SARM GSK2881078 and reveal its illicit use in competitive sports.

Lactate Mediates the Bone Anabolic Effect of High-Intensity Interval Training by Inducing Osteoblast Differentiation.

BACKGROUND: High-intensity interval training (HIIT) reportedly improves bone metabolism and increases bone mineral density (BMD). The purpose of the present study was to investigate whether lactate mediates the beneficial effects of exercise on BMD, bone microarchitecture, and biomechanical properties in an established osteoporotic animal model. In addition, we hypothesized that lactate-induced bone augmentation is achieved through enhanced osteoblast differentiation and mineralization. METHODS: A total of 50 female C57BL/6 mice were randomly allocated into 5 groups: the nonovariectomized group, the ovariectomized group (OVX), the HIIT group (OVX + HIIT), the HIIT with lactate transporter inhibition group (OVX + HIIT + INH), and the lactate subcutaneous injection group (OVX + LAC). After 7 weeks of intervention, bone mass, bone strength, and bone formation/resorption processes were evaluated via microcomputed tomography (micro-CT), biomechanical testing, histological analysis, and serum biochemical assays; in vitro studies were performed to explore the bone anabolic effect of lactate at the cellular level. RESULTS: Micro-CT revealed significantly increased BMD in both the OVX + HIIT group (mean difference, 41.03 mg hydroxyapatite [HA]/cm 3 [95% CI, 2.51 to 79.54 mg HA/cm 3 ]; p = 0.029) and the OVX + LAC group (mean difference, 40.40 mg HA/cm 3 [95% CI, 4.08 to 76.71 mg HA/cm 3 ]; p = 0.031) compared with the OVX group. Biomechanical testing demonstrated significantly improved mechanical properties in those 2 groups. However, the beneficial effects of exercise on bone microstructure and biomechanics were largely abolished by blocking the lactate transporter. Notably, histological and biochemical results indicated that increased bone formation was responsible for the bone augmentation effects of HIIT and lactate. Cell culture studies showed a marked increase in the expression of osteoblastic markers with lactate treatment, which could be eliminated by blocking the lactate transporter. CONCLUSIONS: Lactate may have mediated the bone anabolic effect of HIIT in osteoporotic mice, which may have resulted from enhanced osteoblast differentiation and mineralization. CLINICAL RELEVANCE: Lactate may mediate the bone anabolic effect of HIIT and serve as a potential inexpensive therapeutic strategy for bone augmentation.

The in vivo metabolism of Jungle Warfare in greyhounds.

Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to 17α-methyltestosterone, containing an additional Δ6 double bond, and is reported to possess notable androgenic activity, raising concerns over the potential for abuse of Jungle Warfare in sport. The in vivo metabolism of Δ6-methyltestosterone in greyhounds was investigated. Urinary phase I (unconjugated) and phase II (glucuronide) metabolites were detected following oral administration using liquid chromatography-mass spectrometry. No phase II sulfate metabolites were detected. The major phase I metabolite was confirmed as 16α,17ß-dihydroxy-17α-methylandrosta-4,6-dien-3-one by comparison with a synthetically-derived reference material. Minor amounts of the parent drug were also confirmed. Glucuronide conjugated metabolites were also observed, but were found to be resistant to hydrolysis using the Escherichia coli ß-glucuronidase enzyme. Qualitative excretion profiles, limits of detection, and extraction recoveries were determined for the parent drug and the major phase I metabolite. These results provide a method for the detection of Jungle Warfare abuse in greyhounds suitable for incorporation into routine screening methods conducted by anti-doping laboratories.

Conditioned medium of amniotic fluid-derived stromal cells exerts a bone anabolic effect by enhancing progenitor population and angiogenesis.

A cell-free approach utilizing the paracrine effects of mesenchymal stromal cells is receiving attention in regenerative medicine. In the present study, we evaluated the effects of a conditioned medium of amniotic fluid-derived stromal cells (AFSC-CM) on bone metabolism. In mice, intraperitoneal injections of AFSC-CM increased bone mass and enhanced bone turnover. The precursor populations of myeloid and mesenchymal lineages, as well as endothelial cells in bone marrow, were also augmented by AFSC-CM administration. In an in vitro culture experiment, AFSC-CM increased osteoclast differentiation of bone marrow-derived macrophages, but had no significant effect on the osteogenic differentiation of preosteoblasts. However, AFSC-CM administration dramatically accelerated the migration and tube formation of endothelial cells, and a cytokine array showed that AFSC-CM contained many angiogenic factors. These results indicate that AFSC-CM exerts a bone anabolic effect by changing the bone marrow microenvironment, including angiogenesis and precursor expansion. Therefore, ameliorating marrow angiogenesis is a potential therapeutic strategy for bone regeneration, for which AFSCs can be a good cellular source.

Nandrolone Supplementation Promotes AMPK Activation and Divergent 18[FDG] PET Brain Connectivity in Adult and Aged Mice.

Decreased anabolic androgen levels are followed by impaired brain energy support and sensing with loss of neural connectivity during physiological aging, providing a neurobiological basis for hormone supplementation. Here, we investigated whether nandrolone decanoate (ND) administration mediates hypothalamic AMPK activation and glucose metabolism, thus affecting metabolic connectivity in brain areas of adult and aged mice. Metabolic interconnected brain areas of rodents can be detected by positron emission tomography using 18FDG-mPET. Albino CF1 mice at 3 and 18 months of age were separated into 4 groups that received daily subcutaneous injections of either ND (15 mg/kg) or vehicle for 15 days. At the in vivo baseline and on the 14th day, brain 18FDG-microPET scans were performed. Hypothalamic pAMPKT172/AMPK protein levels were assessed, and basal mitochondrial respiratory states were evaluated in synaptosomes. A metabolic connectivity network between brain areas was estimated based on 18FDG uptake. We found that ND increased the pAMPKT172/AMPK ratio in both adult and aged mice but increased 18FDG uptake and mitochondrial basal respiration only in adult mice. Furthermore, ND triggered rearrangement in the metabolic connectivity of adult mice and aged mice compared to age-matched controls. Altogether, our findings suggest that ND promotes hypothalamic AMPK activation, and distinct glucose metabolism and metabolic connectivity rearrangements in the brains of adult and aged mice.

Species-independent stimulation of osteogenic differentiation induced by osteoclasts.

The coupling of bone resorption and bone formation is well-recognized in the bone remodeling process, in which osteoblasts and osteoclasts are key players. However, the anabolic effect of human primary osteoclasts has rarely been reported as mouse and cell line derived osteoclasts were mostly used in previous reports. Therefore, a comprehensive comparison of mouse and human osteoclasts and their corresponding functions is needed to study cell-cell interactions between osteoclasts and osteoblasts. Osteoclasts from mouse and human origin were generated, characterized and compared, after which their anabolic effects on the osteogenic differentiation of mouse and human MSCs were assessed. Both murine RAW264.7 derived osteoclasts (mOCs) and primary human osteoclasts (hOCs) derived from buffy coats characteristically displayed multinuclearity, marked integrin ß3 expression and enhanced TRAP activity. Despite comparable cell size, mOCs showed higher osteoclast density (number of osteoclasts per cm2 culture dish) and osteoclast nuclearity (average number of nuclei per osteoclast), but lower TRAP activity compared to hOCs. Culturing primary rat and human bone marrow MSCs with the conditioned medium of mOCs or hOCs showed anabolic effects regarding the osteogenic differentiation of MSCs with superiority of hOCs over mOCs. We conclude that despite morphological and functional differences between mouse and human osteoclasts, their secretory factors evoke similar anabolic effects on MSC osteogenic differentiation.

Androgens, sports, and detection strategies for anabolic drug use.

For decades, anabolic androgenic agents have represented the substance class most frequently observed in doping control samples. They comprise synthetic and pseudoendogenous anabolic androgenic steroids and other, mostly non-steroidal compounds with (presumed) positive effects on muscle mass and function. While exogenous substances can easily be detected by gas/liquid chromatography and mass spectrometry, significantly more complex methodologies including the longitudinal monitoring of individual urinary steroid concentrations/ratios and isotope ratio mass spectrometry are required to provide evidence for the exogenous administration of endogenous compounds. This narrative review summarizes the efforts made within the last 5 years to further improve the detection of anabolic agents in doping control samples. Different approaches such as the identification of novel metabolites and biomarkers, the acquisition of complementary mass spectrometric data, and the development of new analytical strategies were employed to increase method sensitivity and retrospectivity while simultaneously reducing method complexity to facilitate a higher and faster sample throughput.

In vitro characterization of S-23 metabolites produced by human liver microsomes, and subsequent application to urine after a controlled oral administration.

The selective androgen receptor modulators are a recent class of anabolic agents, used to improve athletic performance. Among these molecules, there is (2 S)-N-(4-cyano-3-trifluoromethylphenyl)- 3-(3-fluoro-4-chlorophenoxyl)2-hydroxy-2-methyl-propanamide, commonly known as S-23. This molecule appeared very recently on the doping market. As a result, very few data are available in the literature, and nothing has been published about long-term effects of S-23. The authors focused on the detection of S-23 and its metabolites in human urine, following a single oral administration of approx. 8 mg to a volunteer, using standard ultra-performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-MS/MS), and ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-Q-TOF-MS). To the best of the authors knowledge, this seems to be the first study ever achieved on S-23. In vitro experiment was performed, using human liver microsomes, in order to investigate the potential CYP- and UGT-dependent S-23 metabolites. Four metabolites were produced, which were identified as hydroxy-S-23 (C18H12O4N2ClF4: m/z [M-H-] 431.0423); O-dephenylate-S-23 (C12H10O3N2F3: m/z [M-H-] 287.0647); S-23-glucuronide (C24H20O9N2ClF4: m/z [M-H-] 591.0794) and hydroxy-S-23-glucuronide (C24H20O10N2ClF4: m/z [M-H-] 607.0743). After consumption of S-23, the parent drug was detectable in hydrolyzed urine from 2 h post administration up to 28 days, with concentrations ranging between 0.5 and 93 ng/mL. In the urine, only one of the four metabolites identified in vitro was detected, hydroxy-S-23. This metabolite was detected up to 28 days. It does not seem to increase the window of detection of S-23 as the ratio between hydroxy-S-23 and the parent drug was always lower than 1. Another metabolite, dihydroxy-S-23, not identified in vitro, was identified in the urine of the volunteer. Hair sample, collected one month after the consumption of a single tablet, was negative for S-23 and hydroxy-S-23, with a LOQ at 0.1 pg/mg.

In vitro and in vivo metabolism of the anabolic-androgenic steroid oxandrolone in the horse.

Oxandrolone is an anabolic-androgenic steroid with favourable anabolic to androgenic ratio, making it an effective anabolic agent with less androgenic side effects. Although its metabolism has been studied in humans, its phase I and II metabolism has not been previously reported in the horse. The purpose of this study was to investigate the in vitro metabolism of oxandrolone (using both equine liver microsomes and S9) and in vivo metabolism following oral administration (three daily doses of 50 mg of oxandrolone to a single Thoroughbred horse), using both gas and liquid chromatography-mass spectrometry techniques. The in vitro phase I transformations observed included 16-hydroxylated (two epimers), 17-methyl-hydroxylated and 16-keto metabolites. In addition to parent oxandrolone and these hydroxylated metabolites, the 17-epimer and a 17,17-dimethyl-18-norandrost-13-ene analogue were detected in biological samples following the administration. 16-keto-oxandrolone was only observed in urine. The 16- and 17-methyl-hydroxylated oxandrolone metabolites were predominantly excreted as sulfate conjugates in urine, whereas parent oxandrolone, its epimer and 17,17-dimethyl-18-norandrost-13-ene derivative were found predominantly in the unconjugated urine fraction. The most abundant analyte detected in both plasma and urine was parent oxandrolone. However, the longest detection period using the developed analytical method was provided by 17-hydroxymethyl-oxandrolone in both matrices. The results of this study provided knowledge of how best to detect the use of oxandrolone in regulatory samples.

Identification of equine in vitro metabolites of seven non-steroidal selective androgen receptor modulators for doping control purposes.

Selective androgen receptor modulators, SARMs, are a large class of compounds developed to provide therapeutic anabolic effects with minimal androgenic side effects. A wide range of these compounds are available to purchase online and thus provide the potential for abuse in sports. Knowledge of the metabolism of these compounds is essential to aid their detection in doping control samples. In vitro models allow a quick, cost-effective response where administration studies are yet to be carried out. In this study, the equine phase I metabolism of the non-steroidal SARMs GSK2881078, LGD-2226, LGD-3303, PF-06260414, ACP-105, RAD-140 and S-23 was investigated using equine liver microsomes. Liquid chromatography coupled to a QExactive Orbitrap mass spectrometer allowed identification of metabolites with high resolution and mass accuracy. Three metabolites were identified for both GSK2881078 and LGD-2226, four for LGD-3303 and RAD-140, five for PF-06260414, twelve for ACP-105 and ten for S-23. The equine metabolism of GSK-2881078, LGD-2226, LGD-3303 and PF-06260414 is reported for the first time. Although the equine metabolism of ACP-105, RAD-140 and S-23 has previously been reported, the results obtained in this study have been compared with published data.

The in vivo metabolism of Furazadrol in greyhounds.

Samples of the 'dietary supplement' Furazadrol sourced through the internet have been reported to contain the designer anabolic androgenic steroids [1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol (furazadrol F) and [1',2']isoxazolo[4',3':2,3]-5α-androstan-17ß-ol (isofurazadrol IF). These steroids contain an isoxazole fused to the A-ring and were designed to offer anabolic activity while evading detection, raising concerns over the potential for abuse of this preparation in sports. The metabolism of Furazadrol (F:IF, 10:1) was studied by in vivo methods in greyhounds. Urinary phase II Furazadrol metabolites were detected as glucuronides after a controlled administration. These phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli ß-glucuronidase to afford the corresponding phase I metabolites. Using a library of synthetically derived reference materials, the identities of seven urinary Furazadrol metabolites were confirmed. Major confirmed metabolites were isofurazadrol IF, 4α-hydroxyfurazadrol 4α-HF and 16α-hydroxy oxidised furazadrol 16α-HOF, whereas the minor confirmed metabolites were furazadrol F, 4ß-hydroxyfurazadrol 4ß-HF, 16ß-hydroxyfurazadrol 16ß-HF and 16ß-hydroxy oxidised furazadrol 16ß-HOF. One major hydroxyfurazadrol and two dihydroxyfurazadrol metabolites remained unidentified. Qualitative excretion profiles, limits of detection and extraction recoveries were established for furazadrol F and major confirmed metabolites. These investigations identify the key urinary metabolites of Furazadrol following oral administration, which can be incorporated into routine screening by anti-doping laboratories to aid the regulation of greyhound racing.

Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis.

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1'N-glucuronide and 17-epistanozolol-2'N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards. Examination of excretion study samples shows large detection windows for the phase-II metabolites stanozolol-1'N-glucuronide and 17-epistanozolol-1'N-glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid-phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

An Unanticipated Role for Sphingosine Kinase-2 in Bone and in the Anabolic Effect of Parathyroid Hormone.

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Sphingosine kinase (SPHK) is the rate-limiting enzyme in S1P production and has 2 isoforms. To evaluate the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc-/-) and mice in which the SPHK2 gene was deleted in all tissues (SPHK2-/-). SPHK1-Oc-/- had normal bone mass. By contrast, SPHK2-/- female mice had a 14% lower spinal bone mineral density (BMD; P < 0.01) and males a 22% lower BMD at the same site (P < 0.001). SPHK2-/- and control mice were subsequently treated either with daily parathyroid hormone [PTH](1-34) or vehicle for 29 days. The response to PTH was significantly attenuated in the SPHK2-/-mice. The mean femoral bone volume to total volume fraction (BV/TV) increased by 24.8% in the PTH-treated female control animals vs 10.6% in the vehicle-treated female controls (P < 0.01). In contrast, in the SPHK2-/- female mice the difference in femoral trabecular BV/TV at the end of treatment was not significant (20.5 vs13.3%, PTH vs vehicle, P = NS). The anabolic response to PTH was significantly attenuated in the spine of male SPHK2-/- mice (29.7% vs 23.1%, PTH vs vehicle, in controls, P < 0.05; 26.9% vs 19.5% PTH vs vehicle in SPHK2-/- mice, P = NS). The spine responded normally in the SPHK2-/- female mice. Interestingly, suppression of sclerostin was blunted in the SPHK2-/- mice when those animals were treated with an anabolic PTH regimen. We conclude that SPHK2 has an important role in mediating both normal bone remodeling and the anabolic response to PTH.