Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment).

Aspirin (also known as acetylsalicylic acid) is a drug intended to treat fever, pain, or inflammation. Treatment of moderate to severe cases of COVID-19 using aspirin along with dexamethasone has gained major attention globally in recent times. Thus, the purpose of this study was to use High-Performance Liquid Chromatography (HPLC) to evaluate the in vitro inhibition of CYP3A2 enzyme activity using aspirin in rat liver microsomes (RLMs). In this study, an efficient and sensitive HPLC method was developed using a reversed phase C18 column (X Bridge 4.6 mm × 150 mm, 3.5 µm) at 243 nm using acetonitrile and water (70:30 v/v). The linearity (r2 > 0.999), precision (<15%), accuracy and recovery (80-120%), limit of detection (5.60 µM and 0.06 µM), limit of quantification (16.98 µM and 0.19 µM), and stability of the newly developed method were validated for dexamethasone and 6ß-hydroxydexamethasone, respectively, following International Conference on Harmonization (ICH) guidelines. This method was applied in vitro to measure CYP3A2 activity. The results showed that aspirin competitively inhibits 6ß-hydroxylation (CYP3A2 activity) with an inhibition constant (Ki) = 95.46 µM and the concentration of the inhibitor causing 50% inhibition of original enzyme activity (IC50) = 190.92 µM. This indicated that there is a minimal risk of toxicity when dexamethasone and aspirin are co-administrated and a very low risk of toxicity and drug interaction with drugs that are a substrate for CYP3A2 in healthcare settings.

Potential Effects of Ibuprofen, Remdesivir and Omeprazole on Dexamethasone Metabolism in Control Sprague Dawley Male Rat Liver Microsomes (Drugs Often Used Together Alongside COVID-19 Treatment).

The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body's immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with Ki = 224.981 ± 1.854 µM and IC50 = 230.552 ± 2.020 µM, although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 ± 0.008 µM and IC50 = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug's metabolism in vivo.

Potential Assessment of UGT2B17 Inhibition by Salicylic Acid in Human Supersomes In Vitro.

Glucuronidation is a Phase 2 metabolic pathway responsible for the metabolism and excretion of testosterone to a conjugate testosterone glucuronide. Bioavailability and the rate of anabolic steroid testosterone metabolism can be affected upon UGT glucuronidation enzyme alteration. However, there is a lack of information about the in vitro potential assessment of UGT2B17 inhibition by salicylic acid. The purpose of this study is to investigate if UGT2B17 enzyme activity is inhibited by salicylic acid. A UGT2B17 assay was developed and validated by HPLC using a C18 reversed phase column (SUPELCO 25 cm × 4.6 mm, 5 µm) at 246 nm using a gradient elution mobile phase system: (A) phosphate buffer (0.01 M) at pH = 3.8, (B) HPLC grade acetonitrile and (C) HPLC grade methanol. The UGT2B17 metabolite (testosterone glucuronide) was quantified using human UGT2B17 supersomes by a validated HPLC method. The type of inhibition was determined by Lineweaver-Burk plots. These were constructed from the in vitro inhibition of salicylic acid at different concentration levels. The UGT2B17 assay showed good linearity (R2 > 0.99), acceptable recovery and accuracy (80-120%), good reproducibility and acceptable inter and intra-assay precision (<15%), low detection (6.42 and 2.76 µM) and quantitation limit values (19.46 and 8.38 µM) for testosterone and testosterone glucuronide respectively, according to ICH guidelines. Testosterone and testosterone glucuronide were found to be stable up to 72 h in normal laboratory conditions. Our investigational study showed that salicylic acid uncompetitively inhibited UGT2B17 enzyme activity. Thus, drugs that are substrates for the UGT2B17 enzyme have negligible potential effect of causing interaction with salicylic acid in humans.

Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2E1 Enzyme Activity: Inhibitory Effect of Cytochrome P450 Enzyme CYP2E1 by Salicylic Acid in Rat Liver Microsomes.

Inhibition of cytochrome P450 (CYP) alters the pharmacokinetic parameters of the drug and causes drug-drug interactions. Salicylic acid been used for the treatment of colorectal cancer (CRC) and chemoprevention in recent decades. Thus, the aim of this study was to examine the in vitro inhibitory effect of salicylic acid on CYP2E1 activity in rat liver microsomes (RLMs) using high-performance liquid chromatography (HPLC). High-performance liquid chromatography analysis of a CYP2E1 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 282 nm using 60% H2O, 25% acetonitrile, and 15% methanol as mobile phase. The CYP2E1 assay showed a good linearity (R2 > 0.999), good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80-120%), and low detection (4.972 µM and 1.997 µM) and quantitation limit values (15.068 µM and 6.052 µM), for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Salicylic acid acts as a mixed inhibitor (competitive and non-competitive inhibition), with Ki (inhibition constant) = 83.56 ± 2.730 µM and concentration of inhibitor causing 50% inhibition of original enzyme activity (IC50) exceeding 100 µM (IC50 = 167.12 ± 5.460 µM) for CYP2E1 enzyme activity. Salicylic acid in rats would have both low and high potential to cause toxicity and drug interactions with other drugs that are substrates for CYP2E1.

Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2C11 Enzymes in Rat Liver Microsomes: In Vitro Inhibitory Effect of Salicylic Acid on CYP2C11 Enzyme.

The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%-120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug-drug interactions.

Inhibition of Cytochrome P450 Activities by Extracts of Hyptis verticillata Jacq.: Assessment for Potential HERB-Drug Interactions.

Understanding the potential for adverse drug reactions (ADRs), from herb-drug interactions, is a key aspect of medicinal plant safety, with particular relevance for public health in countries where medicinal plant use is highly prevalent. We undertook an in-depth assessment of extracts of Hyptis verticillata Jacq., via its impact on activities of key cytochrome P450 (CYP) enzymes (CYPs 1A1, 1A2, 1B1, 3A4 and 2D6), its antioxidant properties (determined by DPPH assays) and chemical characterisation (using LC-MS). The dried plant aqueous extract demonstrated potent inhibition of the activities of CYPs 1A1 (7.6 µg/mL), 1A2 (1.9 µg/mL), 1B1 (9.4 µg/mL) and 3A4 (6.8 µg/mL). Further analysis of other crude extracts demonstrated potent inhibition of CYP1A2 activity for a dried plant ethanol extract (1.5 µg/mL), fresh plant ethanol extract (3.9 µg/mL), and moderate activity for a fresh plant aqueous extract (27.8 µg/mL). All four extracts demonstrated strong antioxidant activity, compared to the positive control (ascorbic acid, 1.3 µg/mL), with the dried plant ethanol extract being the most potent (1.6 µg/mL). Analysis of the dried plant aqueous extract confirmed the identity of seven phytochemicals, five lignans and two triterpenes. Individual screening of these phytochemicals against the activity of CYP1A2 identified yatein as a moderate inhibitor (71.9 µM), likely to contribute to the plant extract's potent bioactivity. Further analysis on the impact of this plant on key drug metabolizing enzymes in vivo appears warranted for likely ADRs, as well as furthering development as a potential chemopreventive agent.

Inhibitory Effects of Diclofenac on Steroid Glucuronidation In Vivo Do Not Affect Hair-Based Doping Tests for Stanozolol.

In vitro studies show that diclofenac inhibits enzymatic steroid glucuronidation. This study was designed to investigate the influence of diclofenac on the excretion of stanozolol and 3'-hydroxystanozolol via analyses in hair, blood and urine in vivo in a rat study. Brown Norway rats were administered with stanozolol (weeks 1-3) and diclofenac (weeks 1-6). Weekly assessment of steroid levels in hair was complemented with spot urine and serum tests. Levels of both stanozolol and 3'-hydroxystanozolol steadily increased in hair during stanozolol treatment and decreased post-treatment, but remained readily detectable for 6 weeks. In contrast, compared to control rats, diclofenac significantly reduced urinary excretion of 3'-hydroxystanozolol which was undetectable in most samples. This is the first report of diclofenac altering steroid metabolism in vivo, detrimentally affecting detection in urine, but not in hair, which holds considerable advantages over urinalysis for anti-doping tests.

Analysis of 2-oxothiazolidine-4-carboxylic acid by hydrophilic interaction liquid chromatography: application for ocular delivery using chitosan nanoparticles.

Oxidative damage due to low levels of glutathione (GSH) is one of the main causes of cataract formation. It has been reported that 2-oxothiazolidine-4-carboxylic acid (OTZ), a cysteine prodrug, can increase the cellular level of GSH. Currently, there is no analytical method to separate and quantify OTZ from aqueous humour samples for cataract research. The present study aims to develop and validate a hydrophilic interaction liquid chromatography (HILIC) method for the quantification of OTZ in simulated aqueous humour (SAH). The developed method was validated according to FDA guidelines. Accuracy, precision, selectivity, sensitivity, linearity, lower limit of quantification (LLOQ), lower limit of detection (LLOD) and stability were the parameters assessed in the method validation. The developed method was found to be accurate and precise with LLOQ and LLOD of 200 and 100 ng/mL, respectively; method selectivity was confirmed by the absence of any matrix interference with the analyte peak. The constructed calibration curve was linear in the range of 0.2-10 µg/mL, with a regression coefficient of 0.999. In addition, the OTZ was found to be stable in SAH after three freeze/thaw cycles. Chitosan nanoparticles loaded with OTZ were formulated by the ionic gelation method. The nanoparticles were found to be uniform in shape and well dispersed with average size of 153 nm. The in vitro release of OTZ from the nanoparticles was quantified using the developed analytical method over 96 h. Permeation of OTZ through excised bovine cornea was measured using HILIC. The lag time and the flux were 0.2 h and 3.05 µg/cm(2) h, respectively.

In vitro growth characteristics and volatile sulfur compound production of Solobacterium moorei.

Solobacterium moorei has recently been implicated as a causative agent of halitosis. In vitro experiments to evaluate the role of S. moorei in halitosis have, however, been complicated by a paucity of information on the ideal conditions for culturing this organism. This work aimed to optimize a liquid culture medium for S. moorei, and to determine the growth-curve of the organism. Further, the ability of S. moorei to generate volatile sulfur compounds was investigated and compared quantitatively to other oral anaerobes by an optimized head-space gas chromatography method. Serum-supplementation of standard liquid growth media gave greater growth of S. moorei than non-supplemented broths, with the best medium found to be serum-supplemented tryptone soya broth. S. moorei was able to metabolize cysteine directly to hydrogen sulfide, but was unable to produce methanethiol from methionine. S. moorei produced 2-3 times more hydrogen sulfide (normalized for colony forming units) than Porphyromonas gingivalis and Veillonella dispar, but considerably less than Fusobacterium nucleatum. The study has identified reliable growth conditions for culture of S. moorei, which were employed to show that S. moorei has the requisite biochemistry consistent with a potential role in halitosis.

An observational study reveals that neonatal vitamin D is primarily determined by maternal contributions: implications of a new assay on the roles of vitamin D forms.

BACKGROUND: Vitamin D concentrations during pregnancy are measured to diagnose states of insufficiency or deficiency. The aim of this study is to apply accurate assays of vitamin D forms [single- hydroxylated [25(OH)D2, 25(OH)D3], double-hydroxylated [1α,25(OH)2D2, 1a25(OH)2D3], epimers [3-epi-25(OH)D2, 3-epi-25(OH)D3] in mothers (serum) and neonates (umbilical cord) to i) explore maternal and neonatal vitamin D biodynamics and ii) to identify maternal predictors of neonatal vitamin D concentrations. METHODS: All vitamin D forms were quantified in 60 mother- neonate paired samples by a novel liquid chromatography -mass spectrometry (LC-MS/MS) assay. Maternal characteristics [age, ultraviolet B exposure, dietary vitamin D intake, calcium, phosphorus and parathyroid hormone] were recorded. Hierarchical linear regression was used to predict neonatal 25(OH)D concentrations. RESULTS: Mothers had similar concentrations of 25(OH)D2 and 25(OH)D3 forms compared to neonates (17.9 ± 13.2 vs. 15.9 ± 13.6 ng/mL, p=0.289) with a ratio of 1:3. The epimer concentrations, which contribute approximately 25% to the total vitamin D levels, were similar in mothers and neonates (4.8 ± 7.8 vs. 4.5 ± 4.7 ng/mL, p=0.556). No correlation was observed in mothers between the levels of the circulating form (25OHD3) and its active form. Neonatal 25(OH)D2 was best predicted by maternal characteristics, whereas 25(OH)D3 was strongly associated to maternal vitamin D forms (R²=0.253 vs. 0.076 and R2=0.109 vs. 0.478, respectively). Maternal characteristics explained 12.2% of the neonatal 25(OH)D, maternal 25(OH)D concentrations explained 32.1%, while epimers contributed an additional 11.9%. CONCLUSIONS: By applying a novel highly specific vitamin D assay, the present study is the first to quantify 3-epi-25(OH)D concentrations in mother-newborn pairs. This accurate assay highlights a considerable proportion of vitamin D exists as epimers and a lack of correlation between the circulating and active forms. These results highlight the need for accurate measurements to appraise vitamin D status. Maternal characteristics and circulating forms of vitamin D, along with their epimers explain 56% of neonate vitamin D concentrations. The roles of active and epimer forms in the maternal-neonatal vitamin D relationship warrant further investigation.

High-resolution 1H NMR analysis of continuous and discontinuous thermo-oxidative susceptibility of culinary oils during frying at 180 °C.

Lipid oxidations products (LOPs) are reactive mutagenic and carcinogenic species known to be generated in thermally stressed culinary oils. Mapping the evolution of LOPs in culinary oils exposed to standard frying practices - both continuous and discontinuous thermo-oxidation - at 180 °C is vital to our understanding of these processes, and to the development of scientific solutions for their effective suppression. Modifications in the chemical compositions of the thermo-oxidised oils were analysed using a high-resolution proton nuclear magnetic resonance (1H NMR) technique. Research findings acquired showed that polyunsaturated fatty acid (PUFA)-rich culinary oils were the most susceptible to thermo-oxidation. Consistently, coconut oil, which has a very high saturated fatty acid (SFA) content, was highly resistant to the thermo-oxidative methods employed. Furthermore, continuous thermo-oxidation produced greater substantive changes in the oils evaluated than discontinuous episodes. Indeed, for 120 min thermo-oxidation durations, both continuous and discontinuous methods exerted a unique impact on the contents and levels of aldehydic LOPs formed in the oils. This report exposes daily used culinary oils to thermo-oxidation, and therefore, it permits assessments of their peroxidative susceptibilities. It also serves as a reminder to the scientific community to investigate approaches for suppressing toxic LOPs generation in culinary oils exposed to these processes, most notably those involving their reuse.

Red wine and component flavonoids inhibit UGT2B17 in vitro.

BACKGROUND: The metabolism and excretion of the anabolic steroid testosterone occurs by glucuronidation to the conjugate testosterone glucuronide which is then excreted in urine. Alterations in UGT glucuronidation enzyme activity could alter the rate of testosterone excretion and thus its bioavailability. The aim of this study is to investigate if red wine, a common dietary substance, has an inhibitory effect on UGT2B17. METHODS: Testosterone glucuronidation was assayed using human UGT2B17 supersomes with quantification of unglucuronidated testosterone over time using HPLC with DAD detection. The selected red wine was analyzed using HPLC; and the inhibitory effects of the wine and phenolic components were tested independently in a screening assay. Further analyses were conducted for the strongest inhibitors at physiologically relevant concentrations. Control experiments were conducted to determine the effects of the ethanol on UGT2B17. RESULTS: Over the concentration range of 2 to 8%, the red wine sample inhibited the glucuronidation of testosterone by up to 70% over 2 hours. The ethanol content had no significant effect. Three red wine phenolics, identified by HPLC analyses, also inhibited the enzyme by varying amounts in the order of quercetin (72%), caffeic acid (22%) and gallic acid (9%); using a ratio of phenolic:testosterone of 1:2.5. In contrast p-coumaric acid and chlorogenic acid had no effect on the UGT2B17. The most active phenolic was selected for a detailed study at physiologically relevant concentrations, and quercetin maintained inhibitory activity of 20% at 2 µM despite a ten-fold excess of testosterone. CONCLUSION: This study reports that in an in vitro supersome-based assay, the key steroid-metabolizing enzyme UGT2B17 is inhibited by a number of phenolic dietary substances and therefore may reduce the rate of testosterone glucuronidation in vivo. These results highlight the potential interactions of a number of common dietary compounds on testosterone metabolism. Considering the variety of foodstuffs that contain flavonoids, it is feasible that diet can elevate levels of circulating testosterone through reduction in urinary excretion. These results warrant further investigation and extension to a human trial to delineate the health implications.

Understanding how adherence goals promote adherence behaviours: a repeated measure observational study with HIV seropositive patients.

BACKGROUND: The extent to which patients follow treatments as prescribed is pivotal to treatment success. An exceptionally high level (> 95%) of HIV medication adherence is required to suppress viral replication and protect the immune system and a similarly high level (> 80%) of adherence has also been suggested in order to benefit from prescribed exercise programmes. However, in clinical practice, adherence to both often falls below the desirable level. This project aims to investigate a wide range of psychological and personality factors that may lead to adherence/non-adherence to medical treatment and exercise programmes. METHODS: HIV positive patients who are referred to the physiotherapist-led 10-week exercise programme as part of the standard care are continuously recruited. Data on social cognitive variables (attitude, intention, subjective norms, self-efficacy, and outcome beliefs) about the goal and specific behaviours, selected personality factors, perceived quality of life, physical activity, self-reported adherence and physical assessment are collected at baseline, at the end of the exercise programme and again 3 months later. The project incorporates objective measures of both exercise (attendance log and improvement in physical measures such as improved fitness level, weight loss, improved circumferential anthropometric measures) and medication adherence (verified by non-invasive hair analysis). DISCUSSION: The novelty of this project comes from two key aspects, complemented with objective information on exercise and medication adherence. The project assesses beliefs about both the underlying goal such as following prescribed treatment; and about the specific behaviours such as undertaking the exercise or taking the medication, using both implicit and explicit assessments of patients' beliefs and attitudes. We predict that i) the way people think about the underlying goal of their treatments explains medication and exercise behaviours over and above the effects of the behaviour-specific thinking and ii) the relationship between adherence to exercise and to medical treatment is stronger among those with more favourable views about the goal. Results from this study should identify the key contributing factors to inform subsequent adherence research and afford a more streamlined assessment matrix. The project also aims to inform patient care practices. UK CLINICAL RESEARCH NETWORK REGISTRATION NUMBER: UKCRN 7842.

Nuclear Magnetic Resonance Spectroscopic Analysis of the Evolution of Peroxidation Products Arising from Culinary Oils Exposed to Thermal Oxidation: An Investigation Employing 1H and 1H-1H COSY and TOCSY Techniques.

Scientific warnings on the deleterious health effects exerted by dietary lipid oxidation products (LOPs) present in thermally stressed culinary oils have, to date, not received adequate attention given that there has been an increase in the use and consumption of such oil products in everyday life. In this study, high-resolution 1H nuclear magnetic resonance (NMR) analysis was used to characterize and map chemical modifications to fatty acid (FA) acyl groups and the evolution of LOPs in saturated fatty acid (SFA)-rich ghee, monounsaturated fatty acid (MUFA)-rich groundnut, extra virgin olive, and macadamia oils, along with polyunsaturated fatty acid (PUFA)-rich sesame, corn and walnut oils, which were all thermally stressed at 180 °C, continuously and discontinuously for 300 and 480 min, respectively. Results acquired revealed that PUFA-rich culinary oils were more susceptible to thermo-oxidative stress than the others tested, as expected. However, ghee and macadamia oil both generated only low levels of toxic LOPs, and these results demonstrated a striking similarity. Furthermore, at the 120 min thermo-oxidation time-point, the discontinuous thermo-oxidation episodes produced higher concentrations of aldehydic LOPs than those produced during continuous thermo-oxidation sessions for the same duration. On completion of the thermo-oxidation period, a higher level of triacylglycerol chain degradation, and hence, higher concentrations of aldehydes, were registered in culinary oils thermally stressed continuously over those found in discontinuous thermo-oxidized oils. These findings may be crucial in setting targets and developing scientific methods for the suppression of LOPs in thermo-oxidized oils.

The impact of partial oil substitution and trace metal ions on the evolution of peroxidation products in thermally stressed culinary oils.

Suppressing toxic aldehydic lipid oxidation product (LOP) generation in culinary oils is now considered vital, since the deleterious effects arising from their ingestion are implicated in a wide range of disease conditions. Partial substitution involves the replenishment of thermally-stressed culinary oils with corresponding unheated ones. This technique was tested by employing 10%, 25%, 50%, and 75% (v/v) partial substitutions of coconut, olive, rapeseed, and sunflower oils at 180℃ for a 300 min continuous thermo-oxidation duration. Oil samples were analysed by proton nuclear magnetic resonance (1H NMR) spectroscopy. Trace metal levels, including oxidation-reduction (redox)-active metal ions credited with enhancing cooking oil oxidation were also analysed using inductively coupled plasma-optical emission spectroscopy (ICP-OES). As expected, the degree of oil unsaturation, and the % partial substitutions significantly influenced their susceptibility to thermo-oxidation. In view of the very low polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid (MUFA) contents of coconut oil, both the class and concentrations of evolved LOPs were found to be least affected by this partial substitution process. Aldehydic LOPs were greatly suppressed in partially-substituted rapeseed oil. The % suppression activity of LOPs evaluated for the partially substituted oils were generally high making partial oil substitutions an effective chemical-free method in suppressing LOPs at both industrial and commercial levels. In general, the % partial oil substitutions were directly related to the dilution effect observed for LOPs quantified in the oils. Furthermore, trace metal ion concentrations measured in the culinary oils did not influence the evolution of LOPs in the oils.

Misleading measures in Vitamin D analysis: a novel LC-MS/MS assay to account for epimers and isobars.

BACKGROUND: Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars. METHODS: A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified. RESULTS: The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum. CONCLUSIONS: To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies.

Enhancement of antimicrobial activities of whole and sub-fractionated white tea by addition of copper (II) sulphate and vitamin C against Staphylococcus aureus; a mechanistic approach.

BACKGROUND: Enhancement of antimicrobial plant products e.g. pomegranate extract by copper (II) sulphate is known. Such combinations have applications in various settings, including the identification of novel compositions to study, treat and control infection. METHODS: A combination of white tea (WT) (made allowing 10 minutes infusion time at 100°C) was combined with 4.8 mM copper (II) sulphate and tested for antimicrobial effect on the viability of Staphylococcus aureus NCTC 06571. Comparisons were made with green (GT) and black (BT) teas. A WT sub-fraction (WTF < 1000 Da) was tested with copper (II) sulphate and 4.8 mM vitamin C. pH measurements of samples were taken for controls and to observe any changes due to tea/agent interaction. Catalase was used to investigate hydrogen peroxide release. UV-vis. was used to compare WT and WTF. RESULTS: A 30 minute incubation at room temperature of copper (II) sulphate alone and combined with WT reduced the viability of S. aureus NCTC 06571 by c.a 1 log10 cfu mL-1. GT and BT with copper (II) sulphate negated activity to buffer values. Combined with copper (II) sulphate, vitamin C, WTF and, vitamin C plus WTF all reduced the viability of S. aureus NCTC 06571 by c.a. 3.5 log10 cfu mL-1. Independent experiments showed the results were not due to pH effects. Adding WT or WTF to copper (II) sulphate resulted in increased acidity. Copper (II) sulphate alone and combined with WT required c.a 300 µg mL-1 (final concentration) catalase to restore S. aureus viability, WTF with copper (II) sulphate and added vitamin C required c.a 600 µg mL-1. WT and WTF UV-visible spectra were similar. CONCLUSIONS: WT showed no efficacy in the combinations tested. WTF was enhanced with copper (II) sulphate and further with vitamin C. WT and WTF increased acidity of copper (II) sulphate possibly via the formation of chemical complexes. The difference in WT/WTF absorbance possibly represented substances less concentrated or absent in WTF. Investigations to establish which WTF component/s and in what proportions additives are most effective against target organisms are warranted.

A novel non-destructive technique for qualitative and quantitative measurement of dental erosion in its entirety by porosity and bulk tissue-loss.

OBJECTIVE: To explore the potential of combining non-contact profilometry (NCP) and confocal laser scanning microscopy (CLSM) data to measure the entire erosive process non-destructively and to validate findings using inductively coupled plasma-atomic emission spectroscopy (ICP-AES), scanning electron microscopy (SEM) and surface microhardness (SMH) using the same samples throughout. METHODS: Polished bovine enamel samples (n = 35) were divided into groups (7/group) with similar SMH values. Samples underwent individual erosive challenges (1 % citric acid, pH3.8) for 1, 5, 10, 15 or 30 min under stirring and aliquot extracts were analysed for Ca and P by ICP-AES. SMH was used to measure erosive softening. Profilometry was used to assess bulk volume loss (BVL). Images were captured by SEM. Samples were stained with rhodamine-B (0.1 mM, 24 h) and images captured by CLSM. Image processing was used to determine changes in fluorescent volume for the first 10 µm (ΔFV10) for each enamel sample which were combined with BVL to calculate total lesion volume (TLV). ANOVA, linear regression and Pearson correlation analysis were used where applicable. RESULTS: Surface softening, [Ca], [P], BVL and ΔFV10µm increased with acid erosion duration which were significant by 10 min (P < .01). The Ca:P ratio increased to 1.57 then decreased after 5 min erosion suggesting a sub/surface phase change, which was observed by SEM and CLSM showing significant changes to the enamel surface and subsurface morphology with time. Combination of BVL and ΔFV10 as TLV strengthened the significant correlations with [Ca], [P], and SMH (P < .01). CONCLUSION: This novel combination of CLSM and NCP allows for concurrent non-destructive quantification of the entire erosive process by mineral loss, and qualitatively characterise microstructural changes during dental erosion.

The Role of Polydimethylsiloxane in Suppressing the Evolution of Lipid Oxidation Products in Thermo-Oxidised Sunflower Oil: Influence of Stirring Processes.

Suppressing the evolution of lipid oxidation products (LOPs) in commercially available culinary oils is considered to represent a valuable health-promoting incentive since these agents have cytotoxic and genotoxic properties and have been implicated in the pathogenesis of several chronic disease states. One agent used to suppress LOPs formation is polydimethylsiloxane (PDMS). In this study, proton nuclear magnetic resonance (1H NMR) analysis was employed to evaluating the influence of increasing PDMS concentrations (6.25 × 10-7, 1.0 × 10-5, 0.025, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 ppm) in either stirred or unstirred refined sunflower oil exposed to thermal stressing episodes continuously at 180°C for 300 min with no oil replenishment. Results acquired showed that the extent of blockage of LOPs generation was correlated with increasing concentrations of PDMS. The minimal level of added PDMS required to provide a statistically significant protective role for both stirred and unstirred culinary oils when exposed to high frying temperatures was only 6.25 × 10-7 ppm. Furthermore, stirring at 250 rpm was experimentally determined to reduce the functional role PDMS. This is vital in a real world setting since the boiling process of frying may ultimately reduce the LOPs suppression activity of PDMS.

"Real-World" Evaluation of Lipid Oxidation Products and Trace Metals in French Fries From Two Chain Fast-Food Restaurants.

Differences in lipid oxidation products (LOPs) and trace metal concentrations of French fry samples found between two global chain fast-food restaurants in the UK were investigated using high-resolution proton nuclear magnetic resonance (1H NMR) and inductively coupled plasma-optical emission spectrometry (ICP-OES) analyses, respectively, of extracts derived therefrom. Over the course of 3 days and 3 different diurnal time periods, samples of French fries (FFs) were analyzed, and comparisons of two different oil extraction methods were undertaken for the two restaurants involved. The magnitude of concentrations of LOPs extracted from FFs is discussed. Significant differences between 6/7 aldehyde classifications, and aluminum, manganese, vanadium, lead, iron, copper and nickel levels between samples from the two restaurants are also reported. Redox-active transition and further trace metal concentrations inversely correlated with FF oil sample LOP contents; this suggested an antioxidant rather than a pro-oxidant role for them.