Structurally-related (−)-epicatechin metabolites in humans: assessment using de novo chemically synthesized authentic standards

Accumulating data suggest that diets rich in flavanols and procyanidins are beneficial for human health. In this context, there has been a great interest in elucidating the systemic levels and metabolic profiles at which these compounds occur in humans. While recent progress has been made, there still exist considerable differences and various disagreements with regard to the mammalian metabolites of these compounds, which in turn is largely a consequence of the lack of availability of authentic standards that would allow for the directed development and validation of expedient analytical methodologies. In the present study, we developed a method for the analysis of structurally-related flavanol metabolites using a wide range of authentic standards. Applying this method in the context of a human dietary intervention study using comprehensively characterized and standardized flavanol- and procyanidin-containing cocoa, we were able to identify the structurally-related (−)-epicatechin metabolites (SREM) postprandially extant in the systemic circulation of humans. Our results demonstrate that (−)-epicatechin-3′-β-D-glucuronide, (−)-epicatechin-3′-sulfate, and a 3′-O-methyl(−)-epicatechin-5/7-sulfate are the predominant SREM in humans, and further confirm the relevance of the stereochemical configuration in the context of flavanol metabolism. In addition, we also identified plausible causes for the previously reported discrepancies regarding flavanol metabolism, consisting to a significant extent of inter-laboratory differences in sample preparation (enzymatic treatment and sample conditioning for HPLC analysis) and detection systems. Thus, these findings may also aid in the establishment of consensus on this topic.

Intracellular or intercellular localization of the polar pathway of penetration across stratum corneum

A pharmacokinetic hypothesis of stratum corneum with two parallel pathways, lipophilic and porous hydrophilic, is not well documented yet. Still questionable is the localization of the pores, and the present experiments were designed to elucidate the contribution of extracellular lipids and intracellular keratin to the structure of this pathway. Percutaneous penetration of baclofen, a model zwitterion, was studied in vitro using human cadaver skin. Aqueous or ethanolic saturated solutions of the drug (Cs = 4.6 and 0.4 mg/ mL, respectively) were applied on the skin that was pretreated with: methanol/chloroform (Me/Ch) or acetone-chloroform (Ac/Ch) (1:1) mixtures, or with these solvents followed by 0.2% solution of sodium lauryl sulfate (SLS). As controls, baclofen penetration through the intact full-thickness skin was determined, and the fluxes were 0.18 ±0.08 and 0.14 ±0.07 µg/cm2/h for aqueous and ethanolic solutions, respectively. When Me/Ch was used for 1 h, an expected increase of the penetration was observed, but the lag time, Tlag, was still nearly 20 h. When the less polar mixture, Ac/Ch, was used, no flux enhancement was observed, and with ethanol as the vehicle, decreased penetration was even noted. No effect on baclofen penetration was observed when SLS was used for 1 h after delipidization of the skin was done with either the Me/Ch or Ac/Ch mixture. The results suggest that the polar pathway may be located intercellularly and comprises aqueous regions surrounded by polar lipids, which create the walls of such microchannels.

A nipple shield delivery system for oral drug delivery to breastfeeding infants : Microbicide delivery to inactivate HIV

A new drug delivery method for infants is presented which incorporates an active pharmaceutical ingredient (API)-loaded insert into a nipple shield delivery system (NSDS). The API is released directly into milk during breastfeeding. This study investigates the feasibility of using the NSDS to deliver the microbicide sodium dodecyl sulfate (SDS), with the goal of preventing mother-to-child transmission (MTCT) of HIV during breastfeeding in low-resource settings, when there is no safer alternative for the infant but to breastfeed. SDS has been previously shown to effectively inactivate HIV in human milk. An apparatus was developed to simulate milk flow through and drug release from a NSDS. Using this apparatus milk was pulsed through a prototype device containing a non-woven fiber insert impregnated with SDS and the microbicide was rapidly released. The total SDS release from inserts ranged from 70 to 100% of the average 0.07 g load within 50 ml (the volume of a typical breastfeed). Human milk spiked with H9/HIVIIIB cells was also passed through the same set-up. Greater than 99% reduction of cell-associated HIV infectivity was achieved in the first 10 ml of milk. This proof of concept study demonstrates efficient drug delivery to breastfeeding infants is achievable using the NSDS.

Evidence for multiple-antibiotic resistance in Campylobacter jejuni not mediated by CmeB or CmeF

An efflux system, CmeABC, in Campylobacter jejuni was previously described, and a second efflux system, CmeDEF, has now been identified. The substrates of CmeDEF include ampicillin, ethidium bromide, acridine, sodium dodecyl sulfate (SDS), deoxycholate, triclosan, and cetrimide, but not ciprofloxacin or erythromycin. C. jejuni NCTC11168 and two efflux pump knockout strains, cmeB::Kan(r) and cmeF::Kan(r), were exposed to 0.5 to 1 mu g of ciprofloxacin/ml in agar plates. All mutants arising from NCTC11168 were resistant to ciprofloxacin but not to other agents and contained a mutation resulting in the replacement of threonine 86 with isoleucine in the quinolone resistance-determining region of GyrA. Mutants with two distinct phenotypes were selected from the efflux pump knockout strains. Mutants with the first phenotype were resistant to ciprofloxacin only and had the same substitution within GyrA as the NCTC11168-derived mutants. Irrespective of the parent strain, mutants with the second phenotype were resistant to ciprofloxacin, chloramphenicol, tetracycline, ethidium bromide, acridine orange, and SDS and had no mutation in gyrA. These mutants expressed levels of the efflux pump genes cmeB and cmeF and the major outer membrane protein gene porA similar to those expressed by the respective parent strains. No mutations were detected in cmeF or cmeB. Accumulation assays revealed that the mutants accumulated lower concentrations of drug. These data suggest the involvement of a non-CmeB or -CmeF efflux pump or reduced uptake conferring multiple-antibiotic resistance, which can be selected after exposure to a fluoroquinolone.

The influence of ozone precursor emissions from four world regions on tropospheric composition and radiative climate forcing

Ozone (O3) precursor emissions influence regional and global climate and air quality through changes in tropospheric O3 and oxidants, which also influence methane (CH4) and sulfate aerosols (SO42−). We examine changes in the tropospheric composition of O3, CH4, SO42− and global net radiative forcing (RF) for 20% reductions in global CH4 burden and in anthropogenic O3 precursor emissions (NOx, NMVOC, and CO) from four regions (East Asia, Europe and Northern Africa, North America, and South Asia) using the Task Force on Hemispheric Transport of Air Pollution Source-Receptor global chemical transport model (CTM) simulations, assessing uncertainty (mean ± 1 standard deviation) across multiple CTMs. We evaluate steady state O3 responses, including long-term feedbacks via CH4. With a radiative transfer model that includes greenhouse gases and the aerosol direct effect, we find that regional NOx reductions produce global, annually averaged positive net RFs (0.2 ± 0.6 to 1.7 ± 2 mWm−2/Tg N yr−1), with some variation among models. Negative net RFs result from reductions in global CH4 (−162.6 ± 2 mWm−2 for a change from 1760 to 1408 ppbv CH4) and regional NMVOC (−0.4 ± 0.2 to −0.7 ± 0.2 mWm−2/Tg C yr−1) and CO emissions (−0.13 ± 0.02 to −0.15 ± 0.02 mWm−2/Tg CO yr−1). Including the effect of O3 on CO2 uptake by vegetation likely makes these net RFs more negative by −1.9 to −5.2 mWm−2/Tg N yr−1, −0.2 to −0.7 mWm−2/Tg C yr−1, and −0.02 to −0.05 mWm−2/Tg CO yr−1. Net RF impacts reflect the distribution of concentration changes, where RF is affected locally by changes in SO42−, regionally to hemispherically by O3, and globally by CH4. Global annual average SO42− responses to oxidant changes range from 0.4 ± 2.6 to −1.9 ± 1.3 Gg for NOx reductions, 0.1 ± 1.2 to −0.9 ± 0.8 Gg for NMVOC reductions, and −0.09 ± 0.5 to −0.9 ± 0.8 Gg for CO reductions, suggesting additional research is needed. The 100-year global warming potentials (GWP100) are calculated for the global CH4 reduction (20.9 ± 3.7 without stratospheric O3 or water vapor, 24.2 ± 4.2 including those components), and for the regional NOx, NMVOC, and CO reductions (−18.7 ± 25.9 to −1.9 ± 8.7 for NOx, 4.8 ± 1.7 to 8.3 ± 1.9 for NMVOC, and 1.5 ± 0.4 to 1.7 ± 0.5 for CO). Variation in GWP100 for NOx, NMVOC, and CO suggests that regionally specific GWPs may be necessary and could support the inclusion of O3 precursors in future policies that address air quality and climate change simultaneously. Both global net RF and GWP100 are more sensitive to NOx and NMVOC reductions from South Asia than the other three regions.

Why is the global warming proceeding much slower than expected?

Upper air observations from radiosondes and microwave satellite instruments does not indicate any global warming during the last 19 years, contrary to surface measurements, where a warming trend is supposedly being found. This result is somewhat difficult to reconcile, since climate model experiments do indicate a reverse trend, namely, that upper tropospheric air should warm faster than the surface. To contribute toward an understanding of this difficulty, we have here undertaken some specific experiments to study the effect on climate due to the decrease in stratospheric ozone and the Mount Pinatubo eruption in 1991. The associated forcing was added to the forcing from greenhouse gases, sulfate aerosols (direct and indirect effect), and tropospheric ozone, which was investigated in a separate series of experiments. Furthermore, we have undertaken an ensemble study in order to explore the natural variability of an advanced climate model exposed to such a forcing over 19 years. The result shows that the reduction of stratospheric ozone cools not only the lower stratosphere but also the troposphere, in particular, the upper and middle part. In the upper troposphere the cooling from stratospheric ozone leads to a significant reduction of greenhouse warming. The modeled stratospheric aerosols from Mount Pinatubo generate a climate response (stratospheric warming and tropospheric cooling) in good agreement with microwave satellite measurements. Finally, analysis of a series of experiments with both stratospheric ozone and the Mount Pinatubo effect shows considerable variability in climate response, suggesting that an evolution having no warming in the period is as likely as another evolution showing modest warming. However, the observed trend of no warming in the midtroposphere and clear warming at the surface is not found in the model simulations.

Transient climate change simulations with a coupled atmosphere–ocean GCM including the tropospheric sulfur cycle

The time-dependent climate response to changing concentrations of greenhouse gases and sulfate aerosols is studied using a coupled general circulation model of the atmosphere and the ocean (ECHAM4/OPYC3). The concentrations of the well-mixed greenhouse gases like CO2, CH4, N2O, and CFCs are prescribed for the past (1860–1990) and projected into the future according to International Panel on Climate Change (IPCC) scenario IS92a. In addition, the space–time distribution of tropospheric ozone is prescribed, and the tropospheric sulfur cycle is calculated within the coupled model using sulfur emissions of the past and projected into the future (IS92a). The radiative impact of the aerosols is considered via both the direct and the indirect (i.e., through cloud albedo) effect. It is shown that the simulated trend in sulfate deposition since the end of the last century is broadly consistent with ice core measurements, and the calculated radiative forcings from preindustrial to present time are within the uncertainty range estimated by IPCC. Three climate perturbation experiments are performed, applying different forcing mechanisms, and the results are compared with those obtained from a 300-yr unforced control experiment. As in previous experiments, the climate response is similar, but weaker, if aerosol effects are included in addition to greenhouse gases. One notable difference to previous experiments is that the strength of the Indian summer monsoon is not fundamentally affected by the inclusion of aerosol effects. Although the monsoon is damped compared to a greenhouse gas only experiment, it is still more vigorous than in the control experiment. This different behavior, compared to previous studies, is the result of the different land–sea distribution of aerosol forcing. Somewhat unexpected, the intensity of the global hydrological cycle becomes weaker in a warmer climate if both direct and indirect aerosol effects are included in addition to the greenhouse gases. This can be related to anomalous net radiative cooling of the earth’s surface through aerosols, which is balanced by reduced turbulent transfer of both sensible and latent heat from the surface to the atmosphere.

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

Microbial−mammalian cometabolites dominate the age-associated urinary metabolic phenotype in Taiwanese and American populations

Understanding the metabolic processes associated with aging is key to developing effective management and treatment strategies for age-related diseases. We investigated the metabolic profiles associated with age in a Taiwanese and an American population. 1H NMR spectral profiles were generated for urine specimens collected from the Taiwanese Social Environment and Biomarkers of Aging Study (SEBAS; n = 857; age 54–91 years) and the Mid-Life in the USA study (MIDUS II; n = 1148; age 35–86 years). Multivariate and univariate linear projection methods revealed some common age-related characteristics in urinary metabolite profiles in the American and Taiwanese populations, as well as some distinctive features. In both cases, two metabolites—4-cresyl sulfate (4CS) and phenylacetylglutamine (PAG)—were positively associated with age. In addition, creatine and β-hydroxy-β-methylbutyrate (HMB) were negatively correlated with age in both populations (p < 4 × 10–6). These age-associated gradients in creatine and HMB reflect decreasing muscle mass with age. The systematic increase in PAG and 4CS was confirmed using ultraperformance liquid chromatography–mass spectrometry (UPLC–MS). Both are products of concerted microbial–mammalian host cometabolism and indicate an age-related association with the balance of host–microbiome metabolism.

A metabolic system-wide characterisation of the pig: a model for human physiology

The pig is a single-stomached omnivorous mammal and is an important model of human disease and nutrition. As such, it is necessary to establish a metabolic framework from which pathology-based variation can be compared. Here, a combination of one and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy (NMR) and high-resolution magic angle spinning (HR-MAS) NMR was used to provide a systems overview of porcine metabolism via characterisation of the urine, serum, liver and kidney metabolomes. The metabolites observed in each of these biological compartments were found to be qualitatively comparable to the metabolic signature of the same biological matrices in humans and rodents. The data were modelled using a combination of principal components analysis and Venn diagram mapping. Urine represented the most metabolically distinct biological compartment studied, with a relatively greater number of NMR detectable metabolites present, many of which are implicated in gut-microbial co-metabolic processes. The major interspecies differences observed were in the phase II conjugation of extra-genomic metabolites; the pig was observed to conjugate p-cresol, a gut microbial metabolite of tyrosine, with glucuronide rather than sulfate as seen in man. These observations are important to note when considering the translatability of experimental data derived from porcine models.

Observations of the eruption of the Sarychev volcano and simulations using the HadGEM2 climate model

In June 2009 the Sarychev volcano located in the Kuril Islands to the northeast of Japan erupted explosively, injecting ash and an estimated 1.2 ± 0.2 Tg of sulfur dioxide into the upper troposphere and lower stratosphere, making it arguably one of the 10 largest stratospheric injections in the last 50 years. During the period immediately after the eruption, we show that the sulfur dioxide (SO2) cloud was clearly detected by retrievals developed for the Infrared Atmospheric Sounding Interferometer (IASI) satellite instrument and that the resultant stratospheric sulfate aerosol was detected by the Optical Spectrograph and Infrared Imaging System (OSIRIS) limb sounder and CALIPSO lidar. Additional surface‐based instrumentation allows assessment of the impact of the eruption on the stratospheric aerosol optical depth. We use a nudged version of the HadGEM2 climate model to investigate how well this state‐of‐the‐science climate model can replicate the distributions of SO2 and sulfate aerosol. The model simulations and OSIRIS measurements suggest that in the Northern Hemisphere the stratospheric aerosol optical depth was enhanced by around a factor of 3 (0.01 at 550 nm), with resultant impacts upon the radiation budget. The simulations indicate that, in the Northern Hemisphere for July 2009, the magnitude of the mean radiative impact from the volcanic aerosols is more than 60% of the direct radiative forcing of all anthropogenic aerosols put together. While the cooling induced by the eruption will likely not be detectable in the observational record, the combination of modeling and measurements would provide an ideal framework for simulating future larger volcanic eruptions.

Natural aerosol direct and indirect radiative effects

Natural aerosol plays a significant role in the Earth’s system due to its ability to alter the radiative balance of the Earth. Here we use a global aerosol microphysics model together with a radiative transfer model to estimate radiative effects for five natural aerosol sources in the present-day atmosphere: dimethyl sulfide (DMS), sea-salt, volcanoes, monoterpenes, and wildfires. We calculate large annual global mean aerosol direct and cloud albedo effects especially for DMS-derived sulfate (–0.23 Wm–2 and –0.76 Wm–2, respectively), volcanic sulfate (–0.21 Wm–2 and –0.61 Wm–2) and sea-salt (–0.44 Wm–2 and –0.04 Wm–2). The cloud albedo effect responds nonlinearly to changes in emission source strengths. The natural sources have both markedly different radiative efficiencies and indirect/direct radiative effect ratios. Aerosol sources that contribute a large number of small particles (DMS-derived and volcanic sulfate) are highly effective at influencing cloud albedo per unit of aerosol mass burden.

General circulation model estimates of aerosol transport and radiative forcing during the Indian Ocean Experiment

Aerosol sources, transport, and sinks are simulated, and aerosol direct radiative effects are assessed over the Indian Ocean for the Indian Ocean Experiment (INDOEX) Intensive Field Phase during January to March 1999 using the Laboratoire de Me´te´orologie Dynamique (LMDZT) general circulation model. The model reproduces the latitudinal gradient in aerosol mass concentration and optical depth (AOD). The model-predicted aerosol concentrations and AODs agree reasonably well with measurements but are systematically underestimated during high-pollution episodes, especially in the month of March. The largest aerosol loads are found over southwestern China, the Bay of Bengal, and the Indian subcontinent. Aerosol emissions from the Indian subcontinent are transported into the Indian Ocean through either the west coast or the east coast of India. Over the INDOEX region, carbonaceous aerosols are the largest contributor to the estimated AOD, followed by sulfate, dust, sea salt, and fly ash. During the northeast winter monsoon, natural and anthropogenic aerosols reduce the solar flux reaching the surface by 25 W m�2, leading to 10–15% less insolation at the surface. A doubling of black carbon (BC) emissions from Asia results in an aerosol single-scattering albedo that is much smaller than in situ measurements, reflecting the fact that BC emissions are not underestimated in proportion to other (mostly scattering) aerosol types. South Asia is the dominant contributor to sulfate aerosols over the INDOEX region and accounts for 60–70% of the AOD by sulfate. It is also an important but not the dominant contributor to carbonaceous aerosols over the INDOEX region with a contribution of less than 40% to the AOD by this aerosol species. The presence of elevated plumes brings significant quantities of aerosols to the Indian Ocean that are generated over Africa and Southeast and east Asia.

Estimates of global multicomponent aerosol optical depth and direct radiative perturbation in the Laboratoire de Météorologie Dynamique general circulation model

The global cycle of multicomponent aerosols including sulfate, black carbon (BC),organic matter (OM), mineral dust, and sea salt is simulated in the Laboratoire de Me´te´orologie Dynamique general circulation model (LMDZT GCM). The seasonal open biomass burning emissions for simulation years 2000–2001 are scaled from climatological emissions in proportion to satellite detected fire counts. The emissions of dust and sea salt are parameterized online in the model. The comparison of model-predicted monthly mean aerosol optical depth (AOD) at 500 nm with Aerosol Robotic Network (AERONET) shows good agreement with a correlation coefficient of 0.57(N = 1324) and 76% of data points falling within a factor of 2 deviation. The correlation coefficient for daily mean values drops to 0.49 (N = 23,680). The absorption AOD (ta at 670 nm) estimated in the model is poorly correlated with measurements (r = 0.27, N = 349). It is biased low by 24% as compared to AERONET. The model reproduces the prominent features in the monthly mean AOD retrievals from Moderate Resolution Imaging Spectroradiometer (MODIS). The agreement between the model and MODIS is better over source and outflow regions (i.e., within a factor of 2).There is an underestimation of the model by up to a factor of 3 to 5 over some remote oceans. The largest contribution to global annual average AOD (0.12 at 550 nm) is from sulfate (0.043 or 35%), followed by sea salt (0.027 or 23%), dust (0.026 or 22%),OM (0.021 or 17%), and BC (0.004 or 3%). The atmospheric aerosol absorption is predominantly contributed by BC and is about 3% of the total AOD. The globally and annually averaged shortwave (SW) direct aerosol radiative perturbation (DARP) in clear-sky conditions is �2.17 Wm�2 and is about a factor of 2 larger than in all-sky conditions (�1.04 Wm�2). The net DARP (SW + LW) by all aerosols is �1.46 and �0.59 Wm�2 in clear- and all-sky conditions, respectively. Use of realistic, less absorbing in SW, optical properties for dust results in negative forcing over the dust-dominated regions.

Host model uncertainties in aerosol radiative forcing estimates: results from the AeroCom Prescribed intercomparison study

Simulated multi-model “diversity” in aerosol direct radiative forcing estimates is often perceived as a measure of aerosol uncertainty. However, current models used for aerosol radiative forcing calculations vary considerably in model components relevant for forcing calculations and the associated “host-model uncertainties” are generally convoluted with the actual aerosol uncertainty. In this AeroCom Prescribed intercomparison study we systematically isolate and quantify host model uncertainties on aerosol forcing experiments through prescription of identical aerosol radiative properties in twelve participating models. Even with prescribed aerosol radiative properties, simulated clear-sky and all-sky aerosol radiative forcings show significant diversity. For a purely scattering case with globally constant optical depth of 0.2, the global-mean all-sky top-of-atmosphere radiative forcing is −4.47Wm−2 and the inter-model standard deviation is 0.55Wm−2, corresponding to a relative standard deviation of 12 %. For a case with partially absorbing aerosol with an aerosol optical depth of 0.2 and single scattering albedo of 0.8, the forcing changes to 1.04Wm−2, and the standard deviation increases to 1.01W−2, corresponding to a significant relative standard deviation of 97 %. However, the top-of-atmosphere forcing variability owing to absorption (subtracting the scattering case from the case with scattering and absorption) is low, with absolute (relative) standard deviations of 0.45Wm−2 (8 %) clear-sky and 0.62Wm−2 (11 %) all-sky. Scaling the forcing standard deviation for a purely scattering case to match the sulfate radiative forcing in the Aero- Com Direct Effect experiment demonstrates that host model uncertainties could explain about 36% of the overall sulfate forcing diversity of 0.11Wm−2 in the AeroCom Direct Radiative Effect experiment.