Liver Extracellular Vesicles and Particles Enriched ß-Sitosterol Effectively Promote Liver Regeneration in Mice.

Background: The liver's regenerative capacity allows it to repair itself after injury. Extracellular vesicles and particles (EVPs) in the liver's interstitial space are crucial for signal transduction, metabolism, and immune regulation. Understanding the role and mechanism of liver-derived EVPs in regeneration is significant, particularly after partial hepatectomy, where the mechanisms remain unclear. Methods: A 70% hepatectomy model was established in mice, and EVPs were isolated and characterized using electron microscopy, nanocharacterization, and Western blot analysis. Combined metabolomic and transcriptomic analyses revealed ß-sitosterol enrichment in EVPs and activation of the Hedgehog signaling pathway during regeneration. The role of ß-sitosterol in EVPs on the Hedgehog pathway and its targets were identified using qRT-PCR, Western blot analysis. The regulation of carnitine synthesis by this pathway was determined using a dual luciferase assay. The effect of a ß-sitosterol diet on liver regeneration was verified in mice. Results: After 70% hepatectomy, the liver successfully regenerated without liver failure or death. At 24 hours post-surgery, tissue staining showed transient regeneration-associated steatosis (TRAS), with increased Ki67 positivity at 48 hours. EVPs displayed a spherical lipid bilayer structure with particle sizes of 70-130 nm. CD9, CD63, and CD81 in liver-derived EVPs were confirmed. Transcriptomic and metabolomic analyses showed EVPs supplementation significantly promoted carnitine synthesis and fatty acid oxidation. Tissue staining confirmed accelerated TRAS resolution and enhanced liver regeneration with EVP supplementation. Mass spectrometry identified ß-sitosterol in EVPs, which binds to Smo protein, activating the Hedgehog pathway. This led to the nuclear transport of Gli3, stimulating Setd5 transcription and inducing carnitine synthesis, thereby accelerating fatty acid oxidation. Mice with increased ß-sitosterol intake showed faster TRAS resolution and liver regeneration compared to controls. Conclusion: Liver-derived EVPs promote regeneration after partial hepatectomy. ß-sitosterol from EVPs accelerates fatty acid oxidation and promotes liver regeneration by activating Hedgehog signaling pathway.

Chemical Constitutes from Mycelia of Laetiporus versisporus (Agaricomycetes).

Edible mushrooms, both wild and cultivated, can be seen as healthy functional food. More and more valuable compounds are obtained from mycelia of macromycetes. However, there was limited report about the medicinal fungus Laetiporus versisporus (Lloyd) Imazeki. Herein, L. versisporus was fermented on rice media and the secondary metabolites of mycelia were investigated. In this study, two-step method was used to obtain fermented products, silica gel column chromatography, recrystallization, medium pressure column chromatography, preparative thin-layer chromatography were applied to separate the chemical constituents. Nine chemical compounds (1-9) including one new triterpenoid acid versisponic acid F were identified by NMR (nuclear magnetic resonance) spectroscopy and MS (mass spectrometry). Seven compounds including monolinoleoyl glycerol, linoleic acid, ergosta-5, 7, 22-triene-3ß-ol, ß-sitosterol, daucosterol, versisponic acid F were isolated for the first time from L. versisporus.

Mucoadhesive Chitosan Composite Sponge as a Carrier for ß-Sitosterol Cubosomes for Thermal Burn Treatment.

Our study aimed to explore the potential of using nanostructured lipid carriers (NLCs) to enhance the topical administration of ß-sitosterol, a bioactive that is poorly soluble in water. Here, we have taken advantage of the unique characteristics that cubosomes have to provide as a drug delivery system. These characteristics include a large surface area, thermal stability, and the capacity to encapsulate molecules that are hydrophobic, amphiphilic, and hydrophilic. The cubosomal formulation was optimized by building a central composite design. The optimum dispersion exhibited a particle size of 88.3 nm, a zeta potential of -43, a polydispersity index of 0.358, and drug entrapment of 95.6%. It was composed of 15% w/w oleic acid and 5% w/w pluronic F127. The optimized cubosome dispersion was incorporated into a sponge formulation. The optimized cubosome sponge achieved a higher drug release compared with the cubosome dispersion. The SEM micrograph of the selected sponge showed that it has an interwoven irregular fibrous lamellar structure with low density and high porosity. The in-vivo data revealed that topical application of the ß-sitosterol cubosomal sponge showed significant higher wound closure percentage relative to the ß-sitosterol product (Mebo)®.

Synthesis of a Side Chain Alkyne Analogue of Sitosterol as a Chemical Probe for Imaging in Plant Cells.

Clickable chemical tools are essential for studying the localization and role of biomolecules in living cells. For this purpose, alkyne-based close analogs of the respective biomolecules are of outstanding interest. Here, in the field of phytosterols, we present the first alkyne derivative of sitosterol, which fulfills the crucial requirements for such a chemical tool as follows: very similar in size and lipophilicity to the plant phytosterols, and correct absolute configuration at C-24. The alkyne sitosterol FB-DJ-1 was synthesized, starting from stigmasterol, which comprised nine steps, utilizing a novel alkyne activation method, a Johnson-Claisen rearrangement for the stereoselective construction of a branched sterol side chain, and a Bestmann-Ohira reaction for the generation of the alkyne moiety.

Controlling release of astaxanthin in ß-sitosterol oleogel-based emulsions via different self-assembled mechanisms and composition of the oleogelators.

In this study, three types of ß-sitosterol-based oleogels (ß-sitosterol + Î³-oryzanol oleogels, ß-sitosterol + lecithin, oleogels and ß-sitosterol + monostearate oleogels), loaded with astaxanthin, were employed as the oil phase to create oleogel-based emulsions (SO, SL, and SM) using high-pressure homogenization. The microstructure revealed that fine-scale crystals were dispersed within the oil phase of the droplets in the ß-sitosterol oleogel-based emulsion. The bioaccessibility of astaxanthin was found to be 58.13 %, 51.24 %, 36.57 %, and 45.72 % for SM, SL, SO, and the control group, respectively. Interestingly, the release of fatty acids was positively correlated with the availability of astaxanthin (P = 0.981). Further analysis of FFAs release and kinetics indicated that the structural strength of the oil-phase in the emulsions influenced the degree and rate of lipolysis. Additionally, the micellar fraction analysis suggested that the nature and composition of the oleogelators in SM and SL also impacted lipolysis and the bioaccessibility of astaxanthin. Furthermore, interfacial binding of lipase and isothermal titration calorimetry (ITC) measurements revealed that the oleogel network within the oil phase of the emulsion acted as a physical barrier, hindering the interaction between lipase and lipid. Overall, ß-sitosterol oleogel-based emulsions offer a versatile platform for delivering hydrophobic molecules, enhancing the bioavailability of active compounds, and achieving sustained release.

Process optimization for microfluidic preparation of liposomes using food-grade components.

This study explores the potential for optimizing a sustainable manufacturing process that maintains the essential characteristics of conventional liposomes using food-grade solvents and components. The focus was comparing the physicochemical, morphological, and interfacial properties of liposomes produced with these food-grade ingredients to those made by conventional methods. It was found that there was no significant difference in particle size (195.87 ± 1.40 nm) and ζ-potential (-45.13 ± 0.65 mV) between liposomes made from food-grade and conventional materials. The manufacturing process for liposomes, utilizing food-grade solvents and components, was optimized through the application of Plackett-Burman design and response surface methodology. This approach helped identify key parameters (soy lecithin, ß-sitosterol, W/O ratio) and their optimal values (3.17 g, 0.25 g, 1:2.59). These findings suggest that it is possible to enhance the use of liposomes as an effective and safe delivery system in the food industry, adhering to the strict guidelines set by regulatory agencies.

A new flavonolignan from milk thistle (Silybum marianum).

A new flavonolignan, sonyamandin (1), along with other known compounds was isolated from the aerial parts and seeds extracts of Silybum marianum (milk thistle) collected from Jordan. The known ones are ursolic acid (2), oleanolic acid (3), maslinic acid (4), oleic acid (5), ß-sitosterol (6), ß-, sitosteryl glucoside (7), apigenin (8), kaempferol-3-O-rhamnoside (9), apigenin-7-O-ß-D-glycoside (10), isosylibin A (11), isosylibin B (12), and silybin B (13). The absolute stereochemistry of 1 was confirmed by 2D NMR and CD analysis.

Potential Antidiabetic Activity of ß-sitosterol from Zingiber roseum Rosc. via Modulation of Peroxisome Proliferator-activated Receptor Gamma (PPARγ).

AIM: To evaluate the antidiabetic potential of ß-sitosterol from Zingiber roseum. BACKGROUND: Diabetes mellitus is a cluster of metabolic disorders, and 90% of diabetic patients are affected with Type II diabetes (DM2). For the treatment of DM2, thiazolidinedione drugs (TZDs) were proposed, but recent studies have shown that TZDs have several detrimental effects, such as weight gain, kidney enlargement (hypertrophy), fluid retention, increased risk of bone fractures, and potential harm to the liver (hepatotoxicity). That is why a new molecule is needed to treat DM2. OBJECTIVE: The current research aimed to assess the efficacy of ß-Sitosterol from methanolic extract of Zingiber roseum in managing diabetes via PPARγ modulation. METHODS: Zingiber roseum was extracted using methanol, and GC-MS was employed to analyze the extract. Through homology modeling, PPARγ structure was predicted. Molecular docking, MD simulation, free binding energies, QSAR, ADMET, and bioactivity and toxicity scores were all used during the in-depth computer-based research. RESULTS: Clinically, agonists of synthetic thiazolidinedione (TZDs) have been used therapeutically to treat DM2, but these TZDs are associated with significant risks. Hence, GC-MS identified phytochemicals to search for a new PPAR-γ agonist. Based on the in-silico investigation, ß-sitosterol was found to have a higher binding affinity (-8.9 kcal/mol) than standard drugs. MD simulations and MMGBSA analysis also demonstrated that ß-sitosterol bound to the PPAR-γ active site stably. CONCLUSION: It can be concluded that ß-sitosterol from Z. roseum attenuates Type-II diabetes by modulating PPARγ activity.

Effect of different gelators on the physicochemical properties and microstructure of coconut oleogels.

BACKGROUND: The inherent properties of coconut oil (CO), including its elevated saturated fatty acid content and low melting point, make it suitable for application in plastic fat processing. The present study explores the physicochemical characteristics, micromorphology and oxidative stability of oleogels produced from CO using various gelators [ethylcellulose (EC), ß-sitosterol/γ-oryzanol (PS) and glyceryl monostearate (MG)] to elucidate the formation mechanisms of coconut oleogels (EC-COO, PS-COO and MG-COO). RESULTS: Three oleogel systems exhibited a solid-like behavior, with the formation of crystalline forms dominated by ß and ß'. Among them, PS-COO exhibited enhanced capability with respect to immobilizing liquid oils, resulting in solidification with high oil-binding capacity, moderate hardness and good elasticity. By contrast, MG-COO demonstrated inferior stability compared to PS-COO and EC-COO. Furthermore, MG-COO and PS-COO demonstrated antioxidant properties against CO oxidation, whereas EC-COO exhibited the opposite effect. PS-COO and EC-COO exhibited superior thermodynamic behavior compared to MG-COO. CONCLUSION: Three oleogels based on CO were successfully prepared. The mechanical strength, storage modulus and thermodynamic stability of the CO oleogel exhibited concentration dependence with increasing gelling agent addition. PS-COO demonstrated relatively robust oil-binding capacity and oxidative stability, particularly with a 15% PS addition. This information contributes to a deeper understanding of CO-based oleogels and offers theoretical insights for their application in food products. © 2024 Society of Chemical Industry.

Network pharmacology prediction and molecular docking-based strategy to explore the potential mechanism of Duhuo-Jisheng pair against osteoarthritis.

OBJECTIVE: The Duhuo-Jisheng pair is the ruling herb in Duhuo Jisheng decoction, which is a classic formula first recorded in the preparedness and urgency of the thousand jewels. METHODS: We obtained the primary constituents of Duhuo-Jisheng and their associated protein targets from the TCMSP database. We constructed a composite target network using Cytoscape 3.9.1. To identify potential targets for the treatment of osteoarthritis (OA), we retrieved disease targets from OMIM and GeneCards databases and compared them with the composite targets. We imported the overlapping targets into the STRING database to construct a protein-protein interaction (PPI) network. We also conducted Gene ontology (GO) and KEGG enrichment analyses on the targets. RESULTS: The component target network consisted of numerous nodes and edges. Notably, quercetin, ammidin, and ß-sitosterol were identified as the compounds with high degrees. The PPI network identified tumour necrosis factor (TNF), TP53, and NOS2 as proteins with high degrees. The results of GO and KEGG analyses revealed that the signalling pathways used by DHQJD to treat OA included the NF-κB, PI3K-AKT, and TNF pathways. CONCLUSION: Our study provides insights into the effective components and potential molecular mechanisms of Duhuo-Jisheng in treating OA, thus serving as a reference for further basic research in this field.

Controlled volatile release from ß-sitosterol-based oleogels based on different self-assembly mechanisms.

This study examined how the self-assembly mechanisms of ß-sitosterol-based oleogels influenced the release of volatile compounds. Microscopy, X-ray diffraction (XRD) and small-angle X-ray scattering (SAXS) measurements showed that the three ß-sitosterol-based oleogels (ß-sitosterol + Î³-oryzanol oleogels (SO), ß-sitosterol + lecithin oleogels (SL) and ß-sitosterol + monostearate oleogels (SM)) had significant differences in their microstructures, which were formed via different self-assembly mechanisms. SO exhibited the highest oil binding capacity (OBC), complex modulus (G*) and apparent viscosity. Dynamic and static headspace analyses suggested that network structure of ß-sitosterol-based oleogels affected the release of volatile components. SO showed the strongest retention effect, followed by SL and SM. The release of volatile compounds mainly related to structural strength and compositions of oleogels. These results indicated that ß-sitosterol-based oleogels formed with different self-assembly mechanisms have the potential to serve as effective delivery systems for controlling the release of volatile compounds.

Sitosterolemia.

Sitosterolemia is an inherited metabolic disorder characterized by increased levels of plant sterols, such as sitosterol. This disease is caused by loss-of-function genetic mutations in the ATP-binding cassette (ABC) subfamily G member 5 or member 8 (ABCG5 or ABCG8, respectively), both of which play important roles in the selective excretion of plant sterols from the liver and intestine, leading to a failure to excrete plant sterols. Sitosterolemia, which is currently considered a rare genetic disorder, has been described as a phenocopy of homozygous familial hypercholesterolemia (FH). Typical phenotypes of sitosterolemia, including elevated low-density lipoprotein (LDL) cholesterol, tendon xanthomas, and premature coronary artery disease, overlap those of homozygous FH; however, there are substantial differences between these two diseases in terms of treatments and prognoses. Moreover, it is of note that sitosterolemia appears to be quite underdiagnosed, although accurate diagnosis and appropriate interventions will likely to lead to better prognoses compared with homozygous FH. Unlike cases of homozygous FH, dietary counseling is quite effective in reducing the LDL cholesterol as well as sitosterol of patients with sitosterolemia. In this chapter, we summarize the current understandings of this disease and provide useful tips for the diagnosis as well as better treatment of patients with sitosterolemia.

Building blocks of ß-sitosterol-γ-oryzanol gels revealed by small-angle neutron scattering and real space modelling.

Mixtures of ß-sitosterol and γ-oryzanol form gels in a range of organic solvents. Despite being widely studied, particularly as potential oleogels for food application, details of the intrinsic gel-forming building blocks remain unclear. Small-angle neutron scattering (SANS) combined with solvent contrast variation has been used to evaluate potential structural models. While evidence exists that the building blocks are hollow cylinders (tubules), the simultaneous fitting of twelve contrast-varied SANS data sets indicates that the previously proposed model of double walled tubules is incorrect. Predicted scattering based on real space models provides compelling evidence that the origin of the gelling behaviour is the limited assembly of adjacent tubules to form a space-filling network of fibrils.

Virtual Screening and Molecular Docking to Study the Mechanism of Chinese Medicines in the Treatment of Coronavirus Infection.

BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.

The modulation of PI3K/Akt pathway by 3ß hydroxylup-12-en-28-oic acid isolated from Thymus linearis induces cell death in HCT-116 cells.

The presence of intricate carbon skeletons in natural compounds enhances their bioactivity spectrum with unique modes of action at several targets in various dreadful diseases like cancer. The present study was designed to purify the molecules from Thymus linearis and elucidate their antiproliferative activity. The compounds were isolated from the active methanolic extract of Thymus linearis through column chromatography and characterized by various spectroscopic techniques. Antiproliferative activity of isolated compounds was evaluated using MTT assay on cancer and normal cell lines. Mechanism of cell death was elucidated using flow cytometric, microscopic, and Western blot analysis. Four compounds, Sitosterol, Chrysin, 3ß-hydroxylup-12-en-28-oic acid (3BH), and ß-Sitosterol glycoside, were isolated. Among these, 3BH was most potent antiproliferative agent across all cell lines under study, HCT-116 being the most affected one. 3BH was demonstrated to downregulate PI3Ksubunits (p110α and p85α), downstream pAktSer473 and prompted G1 phase cell cycle arrest. The cell cycle CDK inhibitor p27 and p21 were upregulated with simultaneous downregulation of cyclin D1 and cyclin E in HCT-116 cells. This was accompanied by apoptosis, as depicted by decrease in Bcl-2/Bax ratio, with increase in active caspases-3 and caspase-9, cleavage of PARP-1, the generation of reactive oxygen species (ROS), and the loss of mitochondrial membrane potential. The findings established that 3BH induced cell death in HCT-116 cells by modulating PI3K/Akt signaling axis, impeding cell cycle, and instigating apoptosis.

Bioactive Molecules of Mandarin Seed Oils Diminish Mycotoxin and the Existence of Fungi.

Mandarin is a favorite fruit of the citrus family. Mandarin seeds are considered a source of nontraditional oil obtained from byproduct materials. This investigation aimed to assess the biomolecules of mandarin seeds and evaluated their antimycotic and antimycotoxigenic impact on fungi. Moreover, it evaluated the protective role of mandarin oil against aflatoxin toxicity in cell lines. The two types of extracted oil (fixed and volatile) were ecofriendly. The fatty acid composition, tocopherol, sterols, and carotenoids were determined in the fixed oil, whereas volatiles and phenolics were estimated in the essential oil. A mixture of the two oils was prepared and evaluated for its antimicrobial impact. The reduction effect of this mixture was also investigated to reduce mycotoxin secretion using a simulated experiment. The protective effect of the oil was evaluated using healthy strains of cell lines. Fixed oil was distinguished by the omega fatty acid content (76.24%), lutein was the major carotenoid (504.3 mg/100 g) and it had a high ß-sitosterol content (294.6 mg/100 g). Essential oil contained limonene (66.05%), α-pinene (6.82%), ß-pinene (4.32%), and γ-terpinene (12.31%) in significant amounts, while gallic acid and catechol were recorded as the dominant phenolics. Evaluation of the oil mix for antimicrobial potency reflected a considerable impact against pathogenic bacteria and toxigenic fungi. By its application to the fungal media, this oil mix possessed a capacity for reducing mycotoxin secretion. The oil mix was also shown to have a low cytotoxic effect against healthy strains of cell lines and had potency in reducing the mortality impact of aflatoxin B1 applied to cell lines. These results recommend further study to involve this oil in food safety applications.

Phytosterols: Potential Metabolic Modulators in Neurodegenerative Diseases.

Phytosterols constitute a class of natural products that are an important component of diet and have vast applications in foods, cosmetics, and herbal medicines. With many and diverse isolated structures in nature, they exhibit a broad range of biological and pharmacological activities. Among over 200 types of phytosterols, stigmasterol and ß-sitosterol were ubiquitous in many plant species, exhibiting important aspects of activities related to neurodegenerative diseases. Hence, this mini-review presented an overview of the reported studies on selected phytosterols related to neurodegenerative diseases. It covered the major phytosterols based on biosynthetic considerations, including other phytosterols with significant in vitro and in vivo biological activities.

Identification of Sitogluside as a Potential Skin-Pigmentation-Reducing Agent through Network Pharmacology.

Many traditional Chinese medicines (TCMs) with skin-whitening properties have been recorded in the Ben-Cao-Gang-Mu and in folk prescriptions, and some literature confirms that their extracts do have the potential to inhibit pigmentation. However, no systematic studies have identified the specific regulatory mechanisms of the potential active ingredients. The aim of this study was to screen the ingredients in TCMs that inhibit skin pigmentation through a network pharmacology system and to explore underlying mechanisms. We identified 148 potential active ingredients from 14 TCMs, and based on the average "degree" of the topological parameters, the top five TCMs (Fructus Ligustri Lucidi, Hedysarum multijugum Maxim., Ampelopsis japonica, Pseudobulbus Cremastrae Seu Pleiones, and Paeoniae Radix Alba) that were most likely to cause skin-whitening through anti-inflammatory processes were selected. Sitogluside, the most common ingredient in the top five TCMs, inhibits melanogenesis in human melanoma cells (MNT1) and murine melanoma cells (B16F0) and decreases skin pigmentation in zebrafish. Furthermore, mechanistic research revealed that sitogluside is capable of downregulating tyrosinase (TYR) expression by inhibiting the ERK and p38 pathways and inhibiting TYR activity. These results demonstrate that network pharmacology is an effective tool for the discovery of natural compounds with skin-whitening properties and determination of their possible mechanisms. Sitogluside is a novel skin-whitening active ingredient with dual regulatory effects that inhibit TYR expression and activity.

Imidazole-modified C6 -chitosan derivatives used to extract ß-sitosterol from edible oil samples with a microwave-assisted solid phase extraction method.

ß-Sitosterol is a major bioactive constituent in plants with potent anticancer effects against many human cancer cells, but its bioavailability and therapeutic efficacy are limited by its poor solubility in water. In this study, C6 -imidazole chitosan, C6 -1-methylimidazole chitosan, C6 -1-ethylimidazole chitosan, C6 -1-vinylimidazole chitosan, C6 -1-allylimidazole chitosan, and C6 -1-butylimidazole chitosan were prepared to extract ß-sitosterol from edible oil samples via ultrasonic-assisted solid liquid extraction. The structures and properties of the newly synthesized products were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and elemental analysis. The extraction abilities of the derivatives were tested in the experiment with high-performance liquid chromatography (limit of detection 0.21 µg/g and limit of quantification 0.67 µg/g), and the % relative standard deviation (<3.25%) and recovery values of the prepared chitosan derivatives toward ß-sitosterol (average: 100.20%) were acceptable. The spiked interday and intraday recoveries of ß-sitosterol were 102.60 ± 2.78 and 103.90 ± 3.04%, respectively. The actual amounts of ß-sitosterol extracted from three real samples using C6 -imidazole chitosan according to the solid phase extraction method were 3302.40, 901.70, and 2045.60 mg/kg for corn oil, olive oil, and pea oil, respectively.

A fully reversible 25-hydroxy steroid kinase involved in oxygen-independent cholesterol side-chain oxidation.

The degradation of cholesterol and related steroids by microbes follows fundamentally different strategies in aerobic and anaerobic environments. In anaerobic bacteria, the primary C26 of the isoprenoid side chain is hydroxylated without oxygen via a three-step cascade: (i) water-dependent hydroxylation at the tertiary C25, (ii) ATP-dependent dehydration to form a subterminal alkene, and (iii) water-dependent hydroxylation at the primary C26 to form an allylic alcohol. However, the enzymes involved in the ATP-dependent dehydration have remained unknown. Here, we isolated an ATP-dependent 25-hydroxy-steroid kinase (25-HSK) from the anaerobic bacterium Sterolibacterium denitrificans. This highly active enzyme preferentially phosphorylated the tertiary C25 of steroid alcohols, including metabolites of cholesterol and sitosterol degradation or 25-OH-vitamin D3. Kinetic data were in agreement with a sequential mechanism via a ternary complex. Remarkably, 25-HSK readily catalyzed the formation of γ-(18O)2-ATP from ADP and the C25-(18O)2-phosphoester. The observed full reversibility of 25-HSK with an equilibrium constant below one can be rationalized by an unusual high phosphoryl transfer potential of tertiary steroid C25-phosphoesters, which is ≈20 kJ mol-1 higher than that of standard sugar phosphoesters and even slightly greater than the ß,γ-phosphoanhydride of ATP. In summary, 25-HSK plays an essential role in anaerobic bacterial degradation of zoo- and phytosterols and shows only little similarity to known phosphotransferases.