A Novel LC-MS Based Targeted Metabolomic Approach to Study the Biomarkers of Food Intake.

SCOPE: In this work, an integrated strategy is developed for rapid discovery, precise identification, and automated quantification for the biomarkers of food intake (BFIs) for specific food exposure using an ultra-high-pressure liquid chromatography-high-resolution mass spectrometry (MS) based targeted metabolomics approach. METHODS AND RESULTS: Using whole grain (WG) wheat intake as an example, the combination of paired mass distance networking and parallel reaction monitoring analysis is applied to selectively extract and identify WG metabolites in human urine samples. As a result, a total of 76 wheat phytochemical-derived metabolites, including 17 alkylresorcinol metabolites, 20 benzoxazinoid derivatives, and 39 phenolic acid metabolites are identified. Subsequently, a MS spectral database consisting of the identified metabolites is created by mzVault. The characteristics of identified metabolites from the database are incorporated into the TraceFinder software to establish a quantification platform. Using a standardized urine sample, the authors are able to simultaneously quantify both free and conjugated (sulfate and glucuronide) WG wheat metabolites in real samples without further enzymatic hydrolysis, which is validated by using authentic standards to quantify these metabolites. CONCLUSION: This novel strategy opens the window to study the biomarkers of specific food intake and make it feasible to validate the BFIs in large-scale human studies.

Biomarkers of Whole-Grain and Cereal-Fiber Intake in Human Studies: A Systematic Review of the Available Evidence and Perspectives.

High whole-grain consumption is related to better health outcomes. The specific physiological effect of these compounds is still unrevealed, partly because the accurate estimation of the intake of whole grains from dietary assessments is difficult and prone to bias, due to the complexity of the estimation of the intake by the consumer. A biomarker of whole-grain intake and type of whole-grain intake would be useful for quantifying the exposure to whole-grain intake. In this review, we aim to review the evidence on the potential biomarkers for whole-grain intake in the literature. We conducted a systematic search in Medline, Embase, Web of Science, and the Cochrane database. In total, 39 papers met the inclusion criteria following the PRISMA guidelines and were included. The relative validity, responsiveness, and reproducibility of these markers were assessed for short-, medium-, and long-term exposure as important criteria for the potential use of these biomarkers from a clinical and research perspective. We found three major groups of biomarkers: (1) alkylresorcinol, as well as its homologs and metabolites, assessed in plasma, adipose tissue biopsies, erythrocyte membranes, and urine; (2) avenacosides, assessed in urine samples; and (3) benzoxazinoid-derived phenylacetamide sulfates, assessed in blood and urine samples. The reviewed biomarkers may be used for improved assessment of associations between whole-grain intake and health outcomes.

Quantitative analysis of absorption, metabolism, and excretion of benzoxazinoids in humans after the consumption of high- and low-benzoxazinoid diets with similar contents of cereal dietary fibres: a crossover study.

PURPOSE: Benzoxazinoids (BXs) are a group of wholegrain phytochemicals with potential pharmacological properties; however, limited information exists on their absorption, metabolism, and excretion in humans. The aim of this study was to investigate the dose-dependent uptake and excretion of dietary BXs in a healthy population. METHODS: Blood and urine were collected from 19 healthy participants from a crossover study after a washout, a LOW BX diet or HIGH BX diet, and analysed for 12 BXs and 4 phenoxazinone derivatives. RESULTS: We found that the plasma BX level peaked approximately 3 h after food intake, whereas BXs in urine were present even at 36 h after consuming a meal. No phenoxazinone derivatives could be detected in either plasma or urine. The dominant BX metabolite in both plasma and urine was 2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc), even though 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc) was the major component in the diet. CONCLUSION: The dietary BX treatment correlated well with the plasma and urine levels, illustrating strong dose-dependent BX absorption, which also had a rapid washout, especially from the plasma compartment.

Urinary Biomarkers of Whole Grain Wheat Intake Identified by Non-targeted and Targeted Metabolomics Approaches.

Mounting evidence suggests that whole grain (WG) intake plays an important role in chronic disease prevention. However, numerous human studies have failed to produce clear-cut conclusions on this topic. Here, a combination of non-targeted and targeted metabolomics approaches, together with kinetic studies, was used to investigate biomarkers of WG wheat intake and further explore the diet-disease associations. Via these integrated approaches, forty-one compounds were identified as the most discriminating endogenous metabolites after WG versus refined grain (RG) wheat bread consumption. The corresponding biological assessment of these endogenous changes suggests that, in contrast to RG consumption, WG wheat consumption may facilitate antioxidant defense systems and moderate the risk factors of cancer, cardiovascular diseases, and other chronic diseases. A panel of urinary markers consisting of seven alkylresorcinol metabolites and five benzoxazinoid derivatives as specific biomarkers, as well as five phenolic acid derivatives, was also established to cover multiple time points and longer time periods for correctly and objectively monitoring WG wheat intake. Through these findings, we have established a comprehensive biomarker pool to better assess WG wheat consumption, and to monitor the endogenous changes that are linked to health effects of WG wheat consumption.

Benzoxazinoids in Prostate Cancer Patients after a Rye-Intensive Diet: Methods and Initial Results.

Rye bread contains high amounts of benzoxazinoids, and in vitro studies have shown suppressive effects of selected benzoxazinoids on prostate cancer cells. Thus, research into benzoxazinoids as possible suppressors of prostate cancer is demanded. A pilot study was performed in which ten prostate cancer patients received a rye-enriched diet 1 week prior to prostatectomy. Plasma and urine samples were collected pre- and postintervention. Ten prostate biopsies were obtained from each patient and histologically evaluated. The biopsies exhibited concentrations above the detection limit of seven benzoxazinoids ranging from 0.15 to 10.59 ng/g tissue. An OPLS-DA analysis on histological and plasma concentrations of benzoxazinoids classified the subjects into two clusters. A tendency of higher benzoxazinoid concentrations toward the benign group encourages further investigations. Benzoxazinoids were quantified by an optimized LC-MS/MS method, and matrix effects were evaluated. At low concentrations in biopsy and plasma matrices the matrix effect was concentration-dependent and nonlinear. For the urine samples the general matrix effects were small but patient-dependent.

Model-based evaluation of pulmonary pharmacokinetics in asthmatic and COPD patients after oral olodaterol inhalation.

AIMS: Olodaterol is an orally inhaled ß2 -agonist for treatment of chronic obstructive pulmonary disease (COPD). The aims of this population pharmacokinetic (PK) analysis were: (1) to investigate systemic PK and thereby make inferences about pulmonary PK in asthmatic patients, COPD patients and healthy volunteers, and (2) to assess whether differences in pulmonary efficacy might be expected based on pulmonary PK characteristics. METHODS: Plasma and urine data after olodaterol inhalation were available from six clinical trials comprising 710 patients and healthy volunteers (single and multiple dosing). To investigate the relevance of covariates, full fixed-effect modelling was applied based on a previously developed healthy volunteer systemic disposition model. RESULTS: A pulmonary model with three parallel absorption processes best described PK after inhalation in patients. The pulmonary bioavailable fraction (PBIO) was 48.7% (46.1-51.3%, 95% confidence interval) in asthma, and 53.6% (51.1-56.2%) in COPD. In asthma 87.2% (85.4-88.8%) of PBIO was slowly absorbed with an absorption half-life of 18.5 h (16.3-21.4 h), whereas in COPD 80.1% (78.0-82.2%) was absorbed with a half-life of 37.8 h (31.1-47.8 h). In healthy volunteers absorption was faster, with a half-life of 18.5 h (16.3-21.4 h) of the slowest absorbed process, which characterized 74.6% (69.1-80.2%) of PBIO. CONCLUSIONS: The modelling approach successfully described data after olodaterol inhalation in patients and healthy volunteers. Slow pulmonary absorption was demonstrated both in asthma and COPD. Absorption characteristics after olodaterol inhalation indicated even more beneficial lung targeting in patients compared to healthy volunteers.

In Vivo Profiling and Distribution of Known and Novel Phase I and Phase II Metabolites of Efavirenz in Plasma, Urine, and Cerebrospinal Fluid.

Efavirenz (EFV) is principally metabolized by CYP2B6 to 8-hydroxy-efavirenz (8OH-EFV) and to a lesser extent by CYP2A6 to 7-hydroxy-efavirenz (7OH-EFV). So far, most metabolite profile analyses have been restricted to 8OH-EFV, 7OH-EFV, and EFV-N-glucuronide, even though these metabolites represent a minor percentage of EFV metabolites present in vivo. We have performed a quantitative phase I and II metabolite profile analysis by tandem mass spectrometry of plasma, cerebrospinal fluid (CSF), and urine samples in 71 human immunodeficiency virus patients taking efavirenz, prior to and after enzymatic (glucuronidase and sulfatase) hydrolysis. We have shown that phase II metabolites constitute the major part of the known circulating efavirenz species in humans. The 8OH-EFV-glucuronide (gln) and 8OH-EFV-sulfate (identified for the first time) in humans were found to be 64- and 7-fold higher than the parent 8OH-EFV, respectively. In individuals (n = 67) genotyped for CYP2B6, 2A6, and CYP3A metabolic pathways, 8OH-EFV/EFV ratios in plasma were an index of CYP2B6 phenotypic activity (P < 0.0001), which was also reflected by phase II metabolites 8OH-EFV-glucuronide/EFV and 8OH-EFV-sulfate/EFV ratios. Neither EFV nor 8OH-EFV, nor any other considered metabolites in plasma were associated with an increased risk of central nervous system (CNS) toxicity. In CSF, 8OH-EFV levels were not influenced by CYP2B6 genotypes and did not predict CNS toxicity. The phase II metabolites 8OH-EFV-gln, 8OH-EFV-sulfate, and 7OH-EFV-gln were present in CSF at 2- to 9-fold higher concentrations than 8OH-EFV. The potential contribution of known and previously unreported EFV metabolites in CSF to the neuropsychological effects of efavirenz needs to be further examined in larger cohort studies.

Investigating pulmonary and systemic pharmacokinetics of inhaled olodaterol in healthy volunteers using a population pharmacokinetic approach.

AIMS: Olodaterol, a novel ß2-adrenergic receptor agonist, is a long-acting, once-daily inhaled bronchodilator approved for the treatment of chronic obstructive pulmonary disease. The aim of the present study was to describe the plasma and urine pharmacokinetics of olodaterol after intravenous administration and oral inhalation in healthy volunteers by population pharmacokinetic modelling and thereby to infer its pulmonary fate. METHODS: Plasma and urine data after intravenous administration (0.5-25 µg) and oral inhalation (2.5-70 µg via the Respimat® inhaler) were available from a total of 148 healthy volunteers (single and multiple dosing). A stepwise model building approach was applied, using population pharmacokinetic modelling. Systemic disposition parameters were fixed to estimates obtained from intravenous data when modelling data after inhalation. RESULTS: A pharmacokinetic model, including three depot compartments with associated parallel first-order absorption processes (pulmonary model) on top of a four-compartment body model (systemic disposition model), was found to describe the data the best. The dose reaching the lung (pulmonary bioavailable fraction) was estimated to be 49.4% [95% confidence interval (CI) 46.1, 52.7%] of the dose released from the device. A large proportion of the pulmonary bioavailable fraction [70.1% (95% CI 66.8, 73.3%)] was absorbed with a half-life of 21.8 h (95% CI 19.7, 24.4 h). CONCLUSIONS: The plasma and urine pharmacokinetics of olodaterol after intravenous administration and oral inhalation in healthy volunteers were adequately described. The key finding was that a high proportion of the pulmonary bioavailable fraction had an extended pulmonary residence time. This finding was not expected based on the physicochemical properties of olodaterol.

Olodaterol and vilanterol detection in sport drug testing.

The possibility of the detection of olodaterol and vilanterol, two novel ß2 -agonists, in human urine for the purpose of sport drug testing was investigated. Compounds of interest were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) employing methods commonly used in the World Anti-Doping Agency (WADA) accredited laboratories. For both substances, the respective parent compound was found to be a suitable target analyte for monitoring therapeutic dose administration.

Rapid identification of efavirenz metabolites in rats and humans by ultra high performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry.

An in vivo study of efavirenz metabolites in rats and human patients with ultra high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry combined with MetabolitePilot(MT) software is reported for the first time. Considering the polarity differences between the metabolites, solid-phase extraction and protein precipitation were both applied as a part of the sample preparation method. The structures of the metabolites and their fragment ions were identified or tentatively characterized based on the accurate mass and MS(2) data. As a result, a total of 15 metabolites, including 11 from rat samples and 13 from human samples, were identified or tentatively characterized. Two metabolites and several new metabolism pathways are reported for the first time. This study provides a practical approach for identifying complicated metabolites through the rapid and reliable ultra high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry technique, which could be widely used for the investigation of drug metabolites.

Molecularly imprinted nanoparticles prepared by miniemulsion polymerization as a sorbent for selective extraction and purification of efavirenz from human serum and urine.

A molecularly imprinted polymer (MIP) has been synthesized in order to specifically extract efavirenz from serum and urine by dispersive solid-phase extraction following by HPLC-UV analysis. The imprinted nanoparticles were prepared by miniemulsion polymerization method using efavirenz as template molecule and methacrylic acid as functional monomer. Molecular recognition properties, binding capacity and selectivity of the MIPs were evaluated and the results revealed that the obtained MIPs had high specific retention for efavirenz in aqueous medium. The MIP was used as a molecular sorbent for the separation of efavirenz from human serum and urine. The extraction of efavirenz by MIP coupled with HPLC analysis showed a linear calibration curve in the range of 50-300 µg/L with exellent precisions (3.66% and 4.6% for 100 and 300 µg/L respectively). The limit of detection (LOD) and limit of quantification (LOQ) were determind in serum (17.3 and 57.5 µg/L) and urine (10.6 and 36.2 µg/L). The maximum recoveries for serum and urine samples were found to be 95.2% and 92.7% respectively. Due to the high precision and accuracy, this method may be the UV-HPLC choice with MIP extraction for bioequivalence analysis of efavirenz in serum and urine.

Absorption and metabolic fate of bioactive dietary benzoxazinoids in humans.

SCOPE: Benzoxazinoids, which are natural compounds recently identified in mature whole grain cereals and bakery products, have been suggested to have a range of pharmacological properties and health-protecting effects. There are no published reports concerned with the absorption and metabolism of bioactive benzoxazinoids in humans. METHODS AND RESULTS: The absorption, metabolism, and excretion of ten different dietary benzoxazinoids were examined by LC-MS/MS by analyzing plasma and urine from 20 healthy human volunteers after daily intake of 143 µmol of total benzoxazinoids from rye bread and rye buns. The results showed that 2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc) and its oxidized analog, 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc), were the major circulating benzoxazinoids. After consuming a benzoxazinoid diet for 1 week, morning urine contained eight benzoxazinoids with abundant HBOA-Glc (219 nmol × µmol⁻¹ of creatinine). The sulfate and glucuronide conjugates of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) were detected in plasma and urine, indicating substantial phase II metabolism. Direct absorption of lactam glycosides, the reduction of hydroxamic acid glycosides, glucuronidation, and sulfation were the main mechanisms of the absorption and metabolism of benzoxazinoids. CONCLUSION: These results indicate that following ingestion in healthy humans, a range of unmetabolized bioactive dietary benzoxazinoids and their sulfate and glucuronide conjugates appear in circulation and urine.

Hydroxylated phenylacetamides derived from bioactive benzoxazinoids are bioavailable in humans after habitual consumption of whole grain sourdough rye bread.

SCOPE: Understanding relationships between dietary whole grain and health is hindered by incomplete knowledge of potentially bioactive metabolites derived from these foods. We aimed to discover compounds in urine correlated with changes in amounts of whole grain rye consumption. METHODS AND RESULTS: After a wash-out period, volunteers consumed 48-g whole grain rye foods per day for 4 wk and then doubled their intake for a further 4 wk. Samples of 24-h urines were analyzed by flow infusion electrospray MS followed by supervised multivariate data analysis. Urine samples from participants who reported high intakes of rye flakes, rye pasta, or total whole grain rye products could not be discriminated adequately from their wash-out samples. However, discrimination was seen in urine samples from participants who reported high whole grain sourdough rye bread consumption. Accurate mass analysis of explanatory signals followed by fragmentation identified conjugates of the benzoxazinoid lactam 2-hydroxy-1,4-benzoxazin-3-one and hydroxylated phenyl acetamide derivatives. Statistical validation showed sensitivities of 84-96% and specificities of 70-81% (p values < 0·05) for elevated concentrations of these signals after preferential whole grain sourdough rye bread consumption. CONCLUSION: Several potentially bioactive alkaloids have been identified in humans consuming fermented whole grain sourdough rye bread.

UPLC-QTOF/MS metabolic profiling unveils urinary changes in humans after a whole grain rye versus refined wheat bread intervention.

SCOPE: Non-targeted urine metabolite profiling has not been previously exploited in the field of whole grain (WG) products. WG products, particularly rye, are important elements in a healthy Nordic diet. The aim of this study was to identify novel urinary biomarkers of WG rye bread (RB) intake in a randomised crossover study with RB versus refined wheat bread (WB). METHODS AND RESULTS: UPLC-QTOF/MS metabolite profiling was applied to urine from a 2 × 4 wk crossover intervention with RB versus WB in 20 subjects. Sixteen metabolites were revealed as major contributing biomarkers. The most discriminative metabolite after the cereal intervention was identified as 3-(3,5-dihydroxyphenyl)-1-propanoic acid sulphate, which was excreted to a higher extent after the RB versus WB intervention. Other alkylresorcinol metabolites were identified, as well as enterolactone glucuronide, azelaic acid, 2-aminophenol sulphate and its benzoxazinoid precursor 2,4-dihydroxy-1,4-benzoxazin-3-one. Our study also suggests that nitrogen-containing metabolites are other major markers. However, other methodologies will be needed to elucidate their final structure. CONCLUSION: The present non-targeted metabolite profiling proved to be a useful approach to identify major urine metabolites discriminating RB intake from that of white wheat bread. Once validated these markers could help evaluate compliance to healthy Nordic diets.

Plasma and urine concentrations of bioactive dietary benzoxazinoids and their glucuronidated conjugates in rats fed a rye bread-based diet.

Thorough knowledge of the absorption and metabolism of dietary benzoxazinoids is needed to understand their health-promoting effects. In this study, the fates of these bioactive compounds were examined by LC-MS/MS in plasma, urine, and feces after ingesting a daily dose of 4780 ± 68 nmol benzoxazinoids from rye bread using Wistar rats as a model. HBOA-glc (2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one) was the predominant benzoxazinoid in the plasma (74 ± 27 nmol/L), followed by DIBOA-glc (2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one) and HBOA. The total level of benzoxazinoids in the urine was 1176 ± 66 nmol/d, which corresponds to approximately 25% of the total dietary intake. The urinary benzoxazinoid profile differed from that of plasma with HBOA-glc and DIBOA-glc (647 ± 31 and 466 ± 33 nmol/d, respectively) as the major urinary components. The glucuronide conjugates of HBOA and DIBOA were detected in both the plasma and urine. N-dehydroxylation was found to be a critical step in the absorption of hydroxamic acids. This unprecedented study will trigger future interest in the biological effects of benzoxazinoids in whole grain rye and wheat diets in humans and other animals.

Efavirenz interference in urine screening immunoassays for tetrahydrocannabinol.

BACKGROUND: It has been known for some time that the antiretroviral drug, efavirenz (EFV), cross-reacts in urine immunoassays for tetrahydrocannabinol (THC). Because published studies investigating this phenomenon are limited, cross-reactivity information for several immunoassays is lacking. Reports of possible false-positive THC results from clinicians conducting workplace testing prompted us to investigate cross-reactivity for assays frequently employed in our own setting. In light of the potentially deleterious consequences of misclassification, information about EFV cross-reactivity should be included in product information to facilitate interpretation of results and assay selection. METHODS: Random urine samples from 30 patients on EFV therapy were analysed for THC metabolites by two near-testing devices (THC One Step Marijuana and Rapid Response(®) Drugs of Abuse Test Strips) and two automated immunoassays (Roche Diagnostics Cannabinoids II and Beckman Coulter SYNCHRON(®) Systems THC2). THC confirmatory testing was performed by gas chromatography-mass spectrometry (GC-MS). RESULTS: GC-MS failed to detect THC metabolites in any of the samples, as did three of the four immunoassays. However, the Rapid Response(®) test strips yielded positive results in 28 out of 30 samples, which could be reversed on re-testing after sample pretreatment with glucuronidase. CONCLUSIONS: Our study supports previous findings that interference is attributable to a glucuronidated EFV metabolite. We postulate that cross-reactivity is influenced by the composition of immunogens used to elicit anti-THC antibodies. Since access to such information is restricted, contributions from scientists in the antibody industry may be enlightening.

Pharmacokinetics and safety of tanaproget, a nonsteroidal progesterone receptor agonist, in healthy women.

OBJECTIVE: This study aimed to evaluate the pharmacokinetics, pharmacodynamics and safety of the nonsteroidal progesterone receptor agonist, tanaproget. METHODS: A randomized, double-blind, placebo-controlled, sequential-group study of ascending single doses of tanaproget was conducted in healthy, 25- to 45-year-old women on cycle days 8 to 12. Eight subjects (six active, two placebo) per cohort received a dose of 0.1, 0.3, 1, 3, 7 (+/-high-fat meal) or 15 mg. RESULTS: The maximum concentration (C(max)) of tanaproget occurred approximately 2 to 3 h after administration. The elimination half-life (t(1/2)) ranged from 12 to 30 h, and the oral clearance was approximately 70 L/h. The pharmacokinetics of tanaproget was not noticeably altered with a high-fat meal. All doses of tanaproget decreased cervical mucus scores (using a modified Insler method), indicating poor production and poor quality of cervical mucus. The most frequent treatment-emergent adverse events were vaginal bleeding/spotting, abdominal cramping and vomiting; their incidence was not dose related and most events were mild. CONCLUSIONS: Tanaproget was safe and well tolerated, decreased cervical mucus scores and had a pharmacokinetic profile acceptable for use as a once-daily oral contraceptive.