Decolonising food regulatory frameworks: importance of recognising traditional culture when assessing dietary safety of traditional foods.

As interest in Australian native products continues to grow worldwide, Aboriginal and Torres Strait Islander peoples (First Peoples) are striving to be industry leaders in the production of their traditional foods that are being developed for commercial markets. To successfully gain market approval both within Australia and globally, food regulatory authorities require at least a documented history of safe use to indicate dietary safety. Moreover, many countries also require compositional analysis and safety data to further support their safe human consumption. However, safety data are lacking for many of these traditional food items and the history that surrounds their safe use has rarely been recorded in written form, but rather passed on through cultural practices and language. This review evaluates the suitability of current frameworks for assessing the dietary safety of traditional foods and highlights the food-safety regulatory hurdles currently felt by First Peoples and their businesses attempting to enter the Australian native foods industry. These issues also extend to the requirements of food regulatory authorities around the world, when assessing the market eligibility of traditional food items. Potential solutions to these problems are discussed, including new proposed processes that can be incorporated into the current food regulatory frameworks. Importantly, these proposed processes would allow the dietary risk assessment of traditional foods to be completed in a manner that better accommodates the stories, traditional knowledge and interests of First Peoples, while also meeting the safety data requirements set out by regulatory bodies both within Australia and around the world.

Mechanical, corrosion, nanotribological, and biocompatibility properties of equal channel angular pressed Ti-28Nb-35.4Zr alloys for biomedical applications.

This study systematically investigated the effect of equal channel angular pressing (ECAP) on the microstructure, mechanical, corrosion, nano-tribological properties and biocompatibility of a newly developed ß Ti-28Nb-35.4Zr (hereafter denoted TNZ) alloy. Results indicated that ECAP of the ß TNZ alloy refined its microstructure by forming ultrafine grains without causing stress-induced phase transformation, leading to formation of a single ß phase. The ECAP-processed TNZ alloy exhibited a compressive yield strength of 960 MPa, and high plastic deformation capacity without fracturing under compression loads. Potentiodynamic polarization tests revealed the higher tendency of ECAP-processed TNZ alloys to form passive oxide films on its surface, which exhibited a lower corrosion rate (0.44±0.07 µm/y) in Hanks' balanced salt solution compared to its as-cast counterpart (0.71±0.10 µm/y). Nanotribological testing also revealed higher resistance of the ECAP-processed TNZ alloy to abrasion, wear and scratching, when compared to its as-cast counterpart. Cytocompatibility and cell adhesion assessments of the ECAP-processed TNZ alloys showed a high viability (111%) of human osteoblast-like SaOS2 cells after 7 d of culturing. Moreover, the ECAP-processed TNZ alloy promoted adhesion and spreading of SaOS2 cells, which exhibited growth and proliferation on alloy surfaces. In summary, significantly enhanced mechanical, corrosion, and biological properties of ECAP-processed TNZ alloy advocate its suitability for load-bearing implant applications. STATEMENT OF SIGNIFICANCE: Equal channel angular pressing (ECAP) provides a unique combination of enhanced mechanical and functional properties of materials by optimizing their microstructures and phase transformations. This study investigated the mechanical, nano-tribological, corrosion, and biocompatibility properties of a newly developed ß Ti-28Nb-35.4Zr (TNZ) alloy processed via ECAP. Our findings indicated that ECAP of the ß TNZ alloy refined its microstructure by forming ultrafine grains without causing stress-induced phase transformation. Compared to its as-cast counterpart, ECAP-processed TNZ exhibited significantly enhanced compressive yield strength, plastic deformation capacity, hardness, wear, and corrosion properties. Moreover, in vitro cytocompatibility and cell adhesion studies revealed high cellular viabilities, growth and proliferation of osteoblast-like SaOS2 cells on the ECAP-processed TNZ alloy.

Erratum: Xavier et al. High Maternal Omega-3 Supplementation Dysregulates Body Weight and Leptin in Newborn Male and Female Rats: Implications for Hypothalamic Developmental Programming. Nutrients 2021, 13, 89.

The authors would like to correct a typographical error in their recently published paper [...].

Marine Bile Natural Products as Agonists of the TGR5 Receptor.

Agonism of the G protein-coupled bile acid receptor "Takeda G-protein receptor 5" (TGR5) aids in attenuating cholesterol accumulation due to atherosclerotic progression. Although mammalian bile compounds can activate TGR5, they are generally weak agonists, and more effective compounds need to be identified. In this study, two marine bile compounds (5ß-scymnol and its sulfate) were compared with mammalian bile compounds deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) using an in vitro model of TGR5 agonism. The response profiles of human embryonic kidney 293 cells (HEK293) transfected to overexpress TGR5 (HEK293-TGR5) and incubated with subcytotoxic concentrations of test compounds were compared to nontransfected HEK293 control cells using the specific calcium-binding fluorophore Fura-2AM to measure intracellular calcium [Ca2+]i release. Scymnol and scymnol sulfate caused a sustained increase in [Ca2+]i within TGR5 cells only, which was abolished by a specific inhibitor for Gαq protein (UBO-QIC). Sustained increases in [Ca2+]i were seen in both cell types with DCA exposure; this was unaffected by UBO-QIC, indicating that TGR5 activation was not involved. Exposure to UDCA did not alter [Ca2+]i, suggesting a lack of TGR5 bioactivity. These findings demonstrated that both scymnol and scymnol sulfate are novel agonists of TGR5 receptors, showing therapeutic potential for treating atherosclerosis.

An advanced method for quantitative measurements of cholesterol crystallization.

BACKGROUND: Cholesterol crystallization within an atherosclerotic plaque significantly contributes to the acceleration of plaque rupture - a problematic event due to the current lack of specific treatments to prevent such formations. Modelling this pathogenic process is also difficult due to the lack of suitable experimental models that enable quantitative analysis of crystal formation and bioactivity screening of potential therapeutic compounds. AIM: To develop an in vitro human cell model of cholesterol crystallization combined with an imaging system that incorporates both quantitative analysis and real-time continuous imaging of cholesterol crystal formation. METHODS AND RESULTS: An enhanced in vitro model of cholesterol crystallization was developed through the use of acetylated low-density lipoprotein (AcLDL) and 7-ketocholesterol as agents of foam cell induction within a human THP-1 monocytic cell line. Advanced confocal and polarizing microscopies were incorporated into the model so as to allow for quantitation of cholesterol crystallization, with the lipid-loaded group producing significantly greater numbers of cholesterol crystals than the untreated group. The utility of this system was also demonstrated by investigating the effects of the cholesterol-lowering drug lovastatin and therapeutic bile compound ursodeoxycholic acid (UDCA), showing that these drugs influence different aspects of cholesterol crystal formation. CONCLUSIONS: The in vitro human THP-1 monocyte model of cholesterol crystallization provides an effective and efficient means of quantitating cholesterol crystallization in the pre-clinical stage of research. The model also allows for the screening of potentially therapeutic compounds that may be used in attenuating or preventing cholesterol crystallization.

High Maternal Omega-3 Supplementation Dysregulates Body Weight and Leptin in Newborn Male and Female Rats: Implications for Hypothalamic Developmental Programming.

Maternal diet is critical for offspring development and long-term health. Here we investigated the effects of a poor maternal diet pre-conception and during pregnancy on metabolic outcomes and the developing hypothalamus in male and female offspring at birth. We hypothesised that offspring born to dams fed a diet high in fat and sugar (HFSD) peri-pregnancy will have disrupted metabolic outcomes. We also determined if these HFSD-related effects could be reversed by a shift to a healthier diet post-conception, in particular to a diet high in omega-3 polyunsaturated fatty acids (ω3 PUFAs), since ω3 PUFAs are considered essential for normal neurodevelopment. Unexpectedly, our data show that there are minimal negative effects of maternal HFSD on newborn pups. On the other hand, consumption of an ω3-replete diet during pregnancy altered several developmental parameters. As such, pups born to high-ω3-fed dams weighed less for their length, had reduced circulating leptin, and also displayed sex-specific disruption in the expression of hypothalamic neuropeptides. Collectively, our study shows that maternal intake of a diet rich in ω3 PUFAs during pregnancy may be detrimental for some metabolic developmental outcomes in the offspring. These data indicate the importance of a balanced dietary intake in pregnancy and highlight the need for further research into the impact of maternal ω3 intake on offspring development and long-term health.

Mechanical, corrosion, and biocompatibility properties of Mg-Zr-Sr-Sc alloys for biodegradable implant applications.

Magnesium (Mg) and its alloys are considered promising biodegradable implant materials because of their strength and natural degradation in the human body. However, the high corrosion rate of pure Mg in the physiological environment leads to rapid degradation before adequate bone healing. This mismatch between bone healing and the degradation of Mg implants supports the development of new Mg alloys with the addition of other suitable alloying elements in order to achieve simultaneously high corrosion resistance and desirable mechanical properties. This study systematically investigates the microstructure, mechanical properties, corrosion behavior, and biocompatibility of Mg-based alloys with the addition of different concentrations of scandium (Sc), i.e., Mg-0.6Zr-0.5Sr-xSc (x = 0.5, 1, 2, 3 wt.%). Results indicated that high concentration of Sc in strontium (Sr)-containing Mg alloys can alter their microstructures by suppressing the intermetallic phases along the grain boundaries and improve the corrosion resistance by forming chemically stable Sc oxide layers on the surfaces of the Mg alloys. Cytotoxicity assessment revealed that the Sc containing Mg alloys did not significantly alter the viability of human osteoblast-like SaOS2 cells. This study highlights the advantages of using Sc as an alloying element to simultaneously tune Mg alloys with higher strength and slower degradation. STATEMENT OF SIGNIFICANCE: Rare earth elements such as scandium (Sc) with both a high solid-solubility and strong affinity towards oxygen can improve the mechanical and corrosion properties of magnesium (Mg) alloys. However, the feasibility of Sc-containing Mg alloys as biodegradable implant materials is scarcely reported. This study investigates the effects of different Sc concentrations on the mechanical, corrosion, and biocompatibility properties of Mg-Zr-Sr-Sc alloys. Our findings indicated that the addition of Sc significantly improves the mechanical and corrosion properties of Mg-Zr-Sr alloys. Moreover, in vitro cytotoxicity assessment of the Mg-Zr-Sr-Sc alloys did not show any adverse effects on the viability of osteoblast-like cells.

Bisphosphonate activation of crystallized bioglass scaffolds for enhanced bone formation.

The interplay between bone formation by osteoblasts and bone resorption by osteoclasts has a critical effect on bone remodelling processes, and resultant bone quality. Bone scaffolds combined with anti-resorptive bisphosphonate drugs are a promising approach to achieving bone regeneration. Here, we have examined the synergistic effects of the bisphosphonate alendronate (ALD) coated onto calcium phosphate (CaP) modified, sintered bioactive glass 45S5 (BG) scaffolds, on osteoblast stimulation and osteoclast inhibition. After BG pre-treatment with ALD (10-8 M) for 5 days, human MG-63 osteoblasts displayed increased cellular proliferation and significantly enhanced alkaline phosphatase activity (ALP), in comparison with a non-ALD control BG. In contrast, human THP-1-derived osteoclasts cultured with 10-8 M ALD pretreated BG scaffolds showed a significant decrease in tartrate-resistant acid phosphatase (TRAcP) activity, and morphological changes indicative of functional inhibition, including reduced cell size and disruption of the osteoclast sealing zone (F-actin rings). These findings indicate that ALD-coated BG scaffolds promote osteoblast activity and inhibit osteoclast function to enhance bone formation.

LC-MSMS characterisations of scymnol and oxoscymnol biotransformations in incubation mixtures of rat liver microsomes.

The bile alcohol 5ß-scymnol ([24R]-(+)-5ß-cholestan-3α,7α,12α,24,26,27-hexol) is a therapeutic nutraceutical derived from marine sources, however very little is known about its potential for biotransformation as a xenobiotic in higher vertebrates. In this study, biotransformation products of scymnol catalysed by liver microsomes isolated from normal and streptozotocin (STZ)-treated male Wistar rats were characterised by liquid chromatography-tandem mass spectroscopy (LC-MSMS). In order of increasing polarity relative to the reversed phase sorbent, structural assignments were made for four biotransformation products, namely 3-oxoscymnol (5ß-cholestan-3-one-7α,12α,24,26,27-pentol); 7-oxoscymnol (5ß-cholestan-7-one-3α,12α,24,26,27-pentol); 3ß-scymnol (5ß-cholestan-3ß,7α,12α,24,26,27-hexol) and 6ß-hydroxyscymnol (5ß-cholestan-3α,6ß,7α,12α,24,26,27-heptol). In addition, a total of eight biotransformation products were characterised from microsomal incubations of crude oxoscymnol compounds, namely 7ß-scymnol; 3,12-dioxoscymnol; 3,7-dioxoscymnol; 7,12-dioxoscymnol; 12-oxo-3ß-scymnol; 7-oxo-3ß-scymnol; 6ß-hydroxy-12-oxoscymnol and 6ß-hydroxy-7-oxoscymnol. Collectively, the results indicate hepatic enzyme-catalysed hydroxylation, dehydrogenation and epimerisation reactions on the steroid nucleus of scymnol, and provide an insight into biotransformation pathways for scymnol use as a therapeutic nutraceutical in higher vertebrates.

Effect of pre-treatment of crystallized bioactive glass with cell culture media on structure, degradability, and biocompatibility.

The silicate glass 45S5 Bioglass® (BG) is a potential scaffold material for bone regeneration because of its excellent bioactivity, biocompatibility and ability to form a strong bond with bone tissues, via the formation of an apatite layer on its surface. The evaluation of in vitro bioactivity in physiological body fluids, whilst challenging, can offer some insights for developing the bone-bonding ability of these glasses in vivo. In this study, we investigated the influence of three different cell culture and tissue fluid-like solutions on the dissolution and calcium-phosphate (CaP) based re-precipitation behaviour at the glass-liquid interface. We also examined pre-treatment of BG with these biological solutions, and how its influence on bone-forming MG-63 osteoblastic cell proliferation, viability and adhesion. The biological solutions used in this comparative study were: commercial cell culture medium (DMEM), a DMEM solution without organic components (DML) and a simulated body fluid (SBF), incorporating TRIS-buffer. Incubation of BG in these solutions over 28 days resulted in differences in weight loss, solution pH and ion release, and the development of CaP-based surface layers. XRD and FT-IR analyses showed clear differences in the characteristics of the CaP-based coating layers formed by the different solutions. The interfacial reactivity between the glass and the solutions depended on the composition and properties of the solutions. The formation of the CaP layer occurred more rapidly in SBF due to the presence of TRIS-buffer, which also significantly accelerated glass dissolution, further reducing the BG mass in SBF. MG-63 osteoblasts proliferated and spread more rapidly across the surfaces of all pre-conditioned BG, compared to fresh BG. The experimental results of this work help clarify differences between in vitro bioactivity of BG observed in cell culture solutions and in vivo BG bioactivity.

Realistic Exposure Study Assists Risk Assessments of ZnO Nanoparticle Sunscreens and Allays Safety Concerns.

The findings of a new study by Mohammed et al. show that after repeated hourly or daily topical applications typically used for sunscreens, zinc oxide nanoparticles do not penetrate into the viable epidermis or cause toxicity in human skin. This important study confirms that the known benefits of using zinc oxide nanoparticles in sunscreen clearly outweigh the perceived risks of using nanosunscreens.

Porous 45S5 Bioglass®-based scaffolds using stereolithography: Effect of partial pre-sintering on structural and mechanical properties of scaffolds.

Scaffolds made from 45S5 Bioglass® ceramic (BG) show clinical potential in bone regeneration due to their excellent bioactivity and ability to bond to natural bone tissue. However, porous BG scaffolds are limited by their mechanical integrity and by the substantial volume contractions occurring upon sintering. This study examines stereolithographic (SLA) methods to fabricate mechanically robust and porous Bioglass®-based ceramic scaffolds, with regular and interconnected pore networks and using various computer-aided design architectures. It was found that a diamond-like (DM) architecture gave scaffolds the most controllable results without any observable closed porosity in the fired scaffolds. When the pore dimensions of the DM scaffolds of the same porosity (~60vol%) were decreased from 700 to 400µm, the compressive strength values increased from 3.5 to 6.7MPa. In addition, smaller dimensional shrinkage could be obtained by employing partially pre-sintered bioglass, compared to standard 45S5 Bioglass®. Scaffolds derived from pre-sintered bioglass also showed marginally improved compressive strength.

ZnO nanoparticles and organic chemical UV-filters are equally well tolerated by human immune cells.

An important part of assessing the toxic potential of nanoparticles for specific applications should be the direct comparison of biological activities with those of alternative materials for the same application. Nanoparticulate inorganic ultraviolet (UV) filters, such as zinc oxide (ZnO), are commonly incorporated into transparent sunscreen and cosmetic formulations. However, concerns have been raised about potential unwanted effects, despite their negligible skin penetration and inherent advantages over organic chemical UV-filters. To provide useful application-relevant assessments of their potential hazard with/without UVA co-exposure, we directly compared cytotoxic and immune response profiles of human THP-1 monocytic cells to ZnO nanoparticles (30 nm) with bulk ZnO particulates (200 nm) and five conventional organic chemical UV-filters - butylmethoxydibenzoylmethane (avobenzone), octylmethoxycinnamate, octylsalicylate, homosalate and 4-methylbenzylidene camphor. High exposure concentrations of both organic and particulate UV-filters were required to cause cytotoxicity in monocyte and macrophage cultures after 24 h. Co-exposure with UVA (6.7 J/cm(2)) did not alter cytotoxicity profiles. Particle surface area-based dose responses showed that ZnO NPs were better tolerated than bulk ZnO. Organic and particulate UV-filters increased apoptosis at similar doses. Only particulates increased the generation of reactive oxygen species. Interleukin-8 (IL-8) release was increased by all particulates, avobenzone, homosalate and octylsalicylate. IL-1ß release was only increased in macrophages by exposure to avobenzone and homosalate. In conclusion, direct effects were caused in monocytes and macrophages at similar concentrations of both organic UV-filters and ZnO nanoparticulates - indicating that their intrinsic cytotoxicity is similar. With their lower skin penetration, ZnO nanoparticles are expected to have lower bioactivity when used in sunscreens.

Elucidation of the hepatoprotective moiety of 5ß-scymnol that suppresses paracetamol toxicity in mice.

The shark bile alcohol, 5ß-scymnol, protects mice from the hepatotoxic effects of paracetamol (APAP) overdose. To elucidate the hepatoprotective structural moiety of scymnol, we compared its effect with that of its analogue and natural bile salt, sodium scymnol sulfate, in a clinically relevant model of APAP-induced toxicity. Exposure of healthy male Swiss mice to a toxic overdose of APAP (350 mg/kg, ip) significantly increased serum hepatocellular enzyme activities, decreased hepatocellular glutathione (GSH) levels, and induced severe centrilobular hepatocellular necrosis. Repeated low-dose scymnol (5 mg/kg/day for 7 days, ip) significantly reduced the extent of APAP-induced hepatotoxicity without preventing GSH depletion. Sodium scymnol sulfate, which lacks the tri-hydroxyl-substituted aliphatic side chain of scymnol, failed to reduce the APAP hepatotoxicity or prevent GSH depletion when tested under the same experimental conditions. We conclude that the tri-hydroxyl-substituted aliphatic side chain is the hepatoprotective structural moiety of 5ß-scymnol that suppresses APAP-induced cytotoxicity in mice.

Reducing ZnO nanoparticle cytotoxicity by surface modification.

Nanoparticulate zinc oxide (ZnO) is one of the most widely used engineered nanomaterials and its toxicology has gained considerable recent attention. A key aspect for controlling biological interactions at the nanoscale is understanding the relevant nanoparticle surface chemistry. In this study, we have determined the disposition of ZnO nanoparticles within human immune cells by measurement of total Zn, as well as the proportions of extra- and intracellular dissolved Zn as a function of dose and surface coating. From this mass balance, the intracellular soluble Zn levels showed little difference in regard to dose above a certain minimal level or to different surface coatings. PEGylation of ZnO NPs reduced their cytotoxicity as a result of decreased cellular uptake arising from a minimal protein corona. We conclude that the key role of the surface properties of ZnO NPs in controlling cytotoxicity is to regulate cellular nanoparticle uptake rather than altering either intracellular or extracellular Zn dissolution.

Comparison of UVA-induced ROS and sunscreen nanoparticle-generated ROS in human immune cells.

Oxidative damage to cells and tissues from free radicals induced by ultraviolet (UV) irradiation can be attenuated by sunscreen components, such as ZnO and TiO2 nanoparticles (NPs). Although it is known that reactive oxygen species (ROS) are generated by cells upon exposure to ZnO and TiO2 NPs, it is unknown to what extent the amount generated is altered with UV co-exposure. As it is a critical component for determining the relative risk of these NPs when used in sunscreen formulations, we have investigated ROS generation by these NPs in human THP-1 monocyte immune cells following UVA co-exposure. Whilst the applied UVA dose (6.7 J cm(-2)) did not alter cell viability after 24 h, it induced significant ROS production - causing a 7-fold increase in intracellular peroxide and 3.3-fold increase in mitochondrial superoxide levels after 1 h. However, co-exposure to NPs and UVA generated the same or less ROS than with UVA exposure alone, with the exception of anatase TiO2, which showed significantly increased levels. These findings indicate that ROS generation from nanosunscreens is, in most cases, an insignificant contributor to the overall risk associated with oxidative stress from UVA exposure itself.

Relating cytotoxicity, zinc ions, and reactive oxygen in ZnO nanoparticle-exposed human immune cells.

Although zinc oxide (ZnO) nanoparticles (NPs) have been widely formulated in sunscreens, the relationship between reactive oxygen species (ROS) generation induced by these particles, zinc ions, and cytotoxicity is not clearly understood. This study explores whether these factors can be accurately quantified and related. The study demonstrates a strong correlation between ZnO NP-induced cytotoxicity and free intracellular zinc concentration (R (2) = .945) in human immune cells, indicating a requirement for NP dissolution to precede cytotoxicity. In addition, although direct exposure to ZnO NPs was found to induce cytotoxicity at relatively high concentrations, indirect exposure (via dialysis) was not cytotoxic, even at extremely high concentrations, highlighting a requirement for NP-to-cell contact. Elevated levels of ROS present in NP-exposed cells also correlated to both cytotoxicity and intracellular free zinc. Although the addition of antioxidant was able to reduce ROS, cytotoxicity to ZnO NPs was unaffected, suggesting ROS may be, in part, a result of cytotoxicity rather than a causal factor. This study highlights both the requirement and role of intracellular dissolution of zinc nanomaterials to elicit a cytotoxic response. This response is only partially ROS dependent, and therefore, modification of NP uptake and their intracellular solubility are key components in modulating the bioactivity of ZnO NPs.

Formation of zinc-containing nanoparticles from Zn²âº ions in cell culture media: implications for the nanotoxicology of ZnO.

Zinc ions generate a range of poorly soluble Zn-containing nanoparticles when added to commonly used mammalian cell culture media. The formation of these nanoparticles confounds the use of soluble Zn salts as positive controls during cytotoxicity testing of other Zn-containing nanoparticles, such as ZnO. These nanoprecipitates can either be crystalline or amorphous and vary in composition depending upon the concentration of Zn(II) within the medium. The cytotoxicity and immune system response of these nanoparticles in situ are similar to those of 30 nm ZnO nanoparticles. The low residual level of truly soluble Zn species (taken as species passing through a 2 kDa membrane) in cell culture media with serum is insufficient to elicit any appreciable cytotoxicity. These observations highlight the importance of employing appropriate controls when studying ZnO nanoparticle toxicity and suggest a re-evaluation of the conclusions drawn in some previous cytotoxicity studies.

Independent cytotoxic and inflammatory responses to zinc oxide nanoparticles in human monocytes and macrophages.

Significant public and scientific concerns remain for the use of nanoparticles (NPs) in commercial products, particularly those applied topically for skin care. There are currently a range of metal oxides formulated into many sunscreens that are present at the nanoscale. In this study, we sought to determine the effect of the size and dispersion of one type of these NPs (zinc oxide) on immune cell function and cytotoxicity for human macrophages and monocytes, which are key cells for particle and debris clearance in the skin. We have found that particle size and coating, but surprisingly, not agglomeration, are key determinates of nanoparticle cytotoxicity in an in vitro culture system of human immune cells. Most importantly, we found that this nanoparticle-induced cellular immune signalling, can be decoupled from cytotoxicity and surface coating, so that at an equivalent cytotoxic load, smaller particles induce a greater cellular response.

Hydroxysteroid dehydrogenase transformations of 5ß-scymnol and identification of oxoscymnol transformation products by liquid chromatography-tandem mass spectroscopy.

A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS(2)) detection was developed to identify for the first time the oxidation products of 5ß-scymnol [(24R)-(+)-5ß-cholestan-3α,7α,12α,24,26,27-hexol] catalysed by bacterial hydroxysteroid dehydrogenase (HSD) reactions in vitro. The authentic scymnol (MW 468) standard yielded a protonated molecular ion [M+H](+) at m/z 469 Da, and higher mass adduct ions attributed to [M+NH(4)](+) (m/z 486), [M+H+CH(3)OH](+) (m/z 501) and [M+H+CH(3)COOH](+) (m/z 530). (24R)-(+)-5ß-Cholestan-3-one-7α,12α,24,26,27-pentol (3-oxoscymnol, m/z 467 Da, relative retention time (RRT)=0.89) was identified as the principle molecular species of scymnol in the reaction with 3α-HSD pure enzyme. [S](0.5) for the reaction of 3α-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5ß-cholestan-7-one-3α,12α,24,26,27-pentol (7-oxoscymnol, m/z 467 Da, RRT=0.79) and (24R)-(+)-5ß-cholestan-12-one-3α,7α,24,26,27-pentol (12-oxoscymnol, m/z 467 Da, RRT=0.81) were similarly identified as principle molecular species in the respective 7α-HSD and 12α-HSD reactions. Polarity of the oxoscymnol species was established as 7-oxoscymnol>12-oxoscymnol>3-oxoscymnol>scymnol (in order from most polar to least polar). Confirmation that 5ß-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated liquid chromatography-mass spectrometry (LC-MS) techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound.