Determination of metal ion content of beverages and estimation of target hazard quotients : a comparative study

Background: Considerable research has been directed towards the roles of metal ions in nutrition with metal ion toxicity attracting particular attention. The aim of this study is to measure the levels of metal ions found in selected beverages (red wine, stout and apple juice) and to determine their potential detrimental effects via calculation of the Target Hazard Quotients (THQ) for 250 mL daily consumption. Results: The levels (mean ± SEM) and diversity of metals determined by ICP-MS were highest for red wine samples (30 metals totalling 5620.54 ± 123.86 ppb) followed by apple juice (15 metals totalling 1339.87 ± 10.84 ppb) and stout (14 metals totalling 464.85 ± 46.74 ppb). The combined THQ values were determined based upon levels of V, Cr, Mn, Ni, Cu, Zn and Pb which gave red wine samples the highest value (5100.96 ± 118.93 ppb) followed by apple juice (666.44 ± 7.67 ppb) and stout (328.41 ± 42.36 ppb). The THQ values were as follows: apple juice (male 3.11, female 3.87), stout (male 1.84, female 2.19), red wine (male 126.52, female 157.22) and ultra-filtered red wine (male 110.48, female 137.29). Conclusion: This study reports relatively high levels of metal ions in red wine, which give a very high THQ value suggesting potential hazardous exposure over a lifetime for those who consume at least 250 mL daily. In addition to the known hazardous metals (e.g. Pb), many metals (e.g. Rb) have not had their biological effects systematically investigated and hence the impact of sustained ingestion is not known

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

Determination of superoxide dismutase mimetic activity in common culinary herbs

BACKGROUND: Under conditions of oxidative stress, the removal of superoxide, a free radical associated with chronic inflammation, is catalysed by superoxide dismutase (SOD). Thus in addition to acting as an antioxidant, SOD may also be utilized as an anti-inflammatory agent. Some plant derived foods have been shown to have SOD mimetic (SODm) activity however it is not known if this activity is possessed by culinary herbs which have previously been shown to possess both antioxidant and anti-inflammatory properties. The aim of the study was to ascertain if the culinary herbs rosemary, sage and thyme possess SODm activity, and to investigate the influence of cooking and digestion on this activity. Transition metal ion content was also determined to establish if it could likely contribute to any SODm activity detected. FINDINGS: All extracts of uncooked (U), cooked (C) and cooked and digested (C&D) herbs were shown to possess SODm activity, which was significantly correlated with previously determined antioxidant and anti-inflammatory activities of these herbs. SODm activity was significantly increased following (C) and (C&D) for rosemary and sage only. The impact of (C) and (C&D) on the SODm for thyme may have been influenced by its transition metal ion content. CONCLUSIONS: SODm activity may contribute to the herbs' antioxidant and anti-inflammatory activities however the source and significance of this activity need to be established

Potentially harmful advantage to athletes: a putative connection between UGT2B17 gene deletion polymorphism and renal disorders with prolonged use of anabolic androgenic steroids

ABSTRACT: BACKGROUND AND OBJECTIVE: With prolonged use of anabolic androgenic steroids (AAS), occasional incidents of renal disorders have been observed. Independently, it has also been established that there are considerable inter-individual and inter-ethnic differences, in particular with reference to the uridine diphosphate-glucuronosyltransferase 2B17 (UGT2B17) gene, in metabolising these compounds. This report postulates the association of deletion polymorphism in the UGT2B17 gene with the occurrence of renal disorders on chronic exposure to AAS. PRESENTATION OF THE HYPOTHESIS: The major deactivation and elimination pathway of AASs is through glucuronide conjugation, chiefly catalyzed by the UGT2B17 enzyme, followed by excretion in urine. Excretion of steroids is affected in individuals with a deletion mutation in the UGT2B17 gene. We hypothesize that UGT2B17 deficient individuals are more vulnerable to developing renal disorders with prolonged use of AAS owing to increases in body mass index and possible direct toxic effects of steroids on the kidneys. Elevated serum levels of biologically active steroids due to inadequate elimination can lead to prolonged muscle build up. An increase in body mass index may cause renal injuries due to sustained elevated glomerular pressure and flow rate. TESTING THE HYPOTHESIS: In the absence of controlled clinical trials in humans, observational studies can be carried out. Real time PCR with allelic discrimination should be employed to examine the prevalence of different UGT2B17 genotypes in patients with impaired renal function and AAS abuse. In individuals with the UGT2B17 deletion polymorphism, blood tests, biofluid analyses, urinalysis, and hair analyses following the administration of an anabolic steroid can be used to determine the fate of the substance once in the body. IMPLICATIONS OF THE HYPOTHESIS: If the hypothesis is upheld, anabolic steroid users with a deletion mutation in the UGT2B17 gene may be exposed to an increased risk of developing renal disorders. In the current detecting - sanctioning anti-doping system, athletes motivated by the potential to evade detection owing to their unique genetic make-up could subject themselves to a serious health consequence. More research on AAS metabolism in the presence of UGT2B17 gene deletion is required. Benefit - harm evaluations in therapeutic use of anabolic steroids should also consider this potential link between UGT2B17 gene deletion polymorphism and renal disorders

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 (r(2) > 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 6β-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 6β-hydroxylation (CYP3A2 activity) with an inhibition constant (Ki) = 95.46 µM and the concentration of the inhibitor causing 50% inhibition of original enzyme activity (IC(50)) = 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 uM and IC50 = 230.552 +/- 2.020 uM, although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 +/- 0.008 uM and IC50 = 45.007 +/- 0.016 uM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 +/- 0.230 uM and IC50 = 78.351 +/- 0.460 uM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2

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 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 &times; 4.6 mm &times; 5 &micro;m) 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 &gt; 0.999). Substrates and metabolites were found to be stable for up to 72 h. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (&lt;15%), acceptable recovery and accuracy (80%&ndash;120%), and low detection (1.3501 &micro;M and 3.2757 &micro;M) and quantitation limit values (4.914 &micro;M and 9.927 &micro;M) for 16&alpha;-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 &plusmn; 2.67 &micro;M (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 &plusmn; 2.67 &micro;M) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug&ndash;drug interactions

HPLC estimation of iothalamate to measure glomerular filtration rate in humans

Glomerular filtration rate (GFR) is usually determined by estimation of iothalamate (IOT) clearance. We have developed and validated an accurate and robust method for the analysis of IOT in human plasma and urine. The mobile phase consisted of methanol and 50 mM sodium phosphate (10:90; v/v). Flow rate was 1.2 mL/min on a C18 reverse phase column, Synergi-hydro (250 × 4.6 mm) 4 µm 80 Å, with an ultraviolet detector set to 254 nm. Acetonitrile was used for the deproteination and extraction of IOT from human plasma and urine. Precision and accuracy were within 15% for IOT in both plasma and urine. The recoveries of IOT in urine and plasma ranged between 93.14% and 114.74 and 96.04–118.38%, respectively. The linear range for urine and plasma assays were 25–1500 and 1–150 µg/mL respectively. The lower limits of detection were 0.5 µg/mL for both urine and plasma, with no interference from plasma and urine matices. This method has been fully validated according to FDA guidelines and the new HPLC assay has been applied to a new formulation of IOT (Conray™ 43), to calculate GFR in healthy volunteers. The new method is simple, less expensive and it would be instrumental in future clinical and pharmacokinetic studies of iothalamate in kidney patients

Determination of diclofenac concentrations in human plasma using a sensitive gas chromatography mass spectrometry method

Background A gas chromatography mass spectrometry (GCMS) method for the determination of diclofenac in human plasma has been developed and validated. Results This method utilizes hexane which is a relatively less toxic extraction solvent compared to heptane and benzene. In addition, phosphoric acid and acetone were added to the samples as deproteination agents, which increased the recovery of diclofenac. These revised processes allow clean extraction and near-quantitative recovery of analyte (approx. 89–95 %). Separation was achieved on a BP-1 column with helium as carrier gas. The molecular ion peaks of the indolinone derivatives of diclofenac ion (m/z 277) and the internal standard, 4-hydroxydiclofenac ion (m/z 439) were monitored by a mass-selective detector using selected ion monitoring (SIM) mode. The linear range for the newly developed and highly sensitive assay was between 0.25–50 ng/mL. The detection and lower quantifiable limits were 0.125 and 0.25 ng/mL, respectively. The inter-day and intra-day coefficients of variation for high, medium and low quality control concentrations were less than 9 %. The robustness and efficacy of this sensitive GCMS method was further demonstrated by using it for a pharmacokinetic study of an oral dosage form of diclofenac, 100 mg of modified-release capsules (Rhumalgan XL), in human plasma. Conclusions This method is rapid, sensitive, specific, reproducible and robust, and offers improved sensitivity over previous methods. This method has considerable potential to be used for detailed pharmacokinetics, pharmacodynamics and bioequivalence studies of diclofenac in humans