Synergic effects of sugar and caffeine on insulin-mediated metabolomic alterations after an acute consumption of soft drinks.

High sugar consumption elicits numerous deleterious effects on health by inducing insulin resistance, which is closely associated with the development of metabolic disorders such as obesity or type-2 diabetes. Furthermore, there is also growing evidence that caffeine may play an important role in the regulation of insulin release and the appearance of related metabolic impairments. Thus, the aim of this work was to investigate the impact of acute sugar and caffeine intake on the metabolic health status by using a metabolomic multi-platform based on the combination of flow injection mass spectrometry and ultra-high performance liquid chromatography mass spectrometry. To this end, we performed a randomized, crossover and double-blind intervention study with different soft drinks from the same brand. Numerous metabolomic changes were detected in serum samples over time after the intake of sugar-sweetened beverages, including energy-related metabolites, amino acids and lipids, thus demonstrating the intense effects provoked by acute sugar consumption on the organism during 3 h of follow-up. However, the most significant findings were observed after the co-ingestion of caffeine, which could be indicative of a synergic effect of this psychostimulant on insulin-mediated perturbations.

Lost opportunities to identify and treat HIV-positive patients: results from a baseline assessment of provider-initiated HIV testing and counselling (PITC) in Malawi.

OBJECTIVE: To assess implementation of provider-initiated testing and counselling (PITC) for HIV in Malawi. METHODS: A review of PITC practices within 118 departments in 12 Ministry of Health (MoH) facilities across Malawi was conducted. Information on PITC practices was collected via a health facility survey. Data describing patient visits and HIV tests were abstracted from routinely collected programme data. RESULTS: Reported PITC practices were highly variable. Most providers practiced symptom-based PITC. Antenatal clinics and maternity wards reported widespread use of routine opt-out PITC. In 2014, there was approximately 1 HIV test for every 15 clinic visits. HIV status was ascertained in 94.3% (5293/5615) of patients at tuberculosis clinics, 92.6% (30,675/33,142) of patients at antenatal clinics and 49.4% (6871/13,914) of patients at sexually transmitted infection clinics. Reported challenges to delivering PITC included test kit shortages (71/71 providers), insufficient physical space (58/71) and inadequate number of HIV counsellors (32/71) while providers from inpatient units cited the inability to test on weekends. CONCLUSIONS: Various models of PITC currently exist at MoH facilities in Malawi. Only antenatal and maternity clinics demonstrated high rates of routine opt-out PITC. The low ratio of facility visits to HIV tests suggests missed opportunities for HIV testing. However, the high proportion of patients at TB and antenatal clinics with known HIV status suggests that routine PITC is feasible. These results underscore the need to develop clear, standardised PITC policy and protocols, and to address obstacles of limited health commodities, infrastructure and human resources.

Aroma of Sherry Products: A Review.

Jerez (Sherry) is a well-known wine-producing region located in southern Spain, where world-renowned oenological products such as wines, vinegars, and brandies are produced. There are several factors that provide characteristic physical, chemical, and sensory properties to the oenological products obtained in this Sherry region: the climate in the area with hot summers, mild winters, and with limited rainfall; the raw material used consisting on Palomino Fino, Moscatel, and Pedro Ximénez white grape varieties; the special vinification with fortified wines; and aging techniques such as a dynamic system of biological or oxidative aging. These special organoleptic characteristics are responsible for, among others, the aromatic profile of the wines, vinegars and brandies from the area, which explains why this is a subject that has been extensively researched over the years. This bibliographic review aims to compile the different scientific contributions that have been found to date, in relation with the aroma of the oenological products from the Sherry area (dry wines, sweet wines, vinegars, and brandies). We have mainly focused on the different analytical methodologies used and on the main analytes of interest.

HPLC-DAD-MS and Antioxidant Profile of Fractions from Amontillado Sherry Wine Obtained Using High-Speed Counter-Current Chromatography.

In the present work, the polyphenolic profile of a complex matrix such as Amontillado sherry has been processed by means of high-speed counter-current chromatography (HSCCC) and characterized by HPLC-DAD-MS. An Amberlite XAD-7 column was used to obtain the wine extract, and three different biphasic solvent systems were applied for HSCCC separation: MTBE (methyl tert-butyl ether)/n-butanol/acetonitrile/water (1.1/3/1.1/5+0.1% trifluoroacetic acid), MTBE/n-butanol/acetonitrile/water (2/2/1/5), and hexane/ethyl acetate/ethanol/water (1/5/1/5). As a result, 42 phenolic compounds and furanic derivatives have been identified by means of HPLC-DAD-MS, with 11 of them being identified for the first time in Sherry wines: 3-feruloylquinic acid, isovanillin, ethyl vanillate, furoic acid, dihydro-p-coumaric acid, 6-O-feruloylglucose, ethyl gallate, hydroxytyrosol, methyl protocatechuate, homoveratric acid and veratraldehyde. In addition, the antioxidant capacity (ABTS) of the obtained fractions was determined, revealing higher values in those fractions in which compounds such as gallic acid, protocatechuic acid, protocatechualdehyde, trans-caftaric acid, syringic acid, isovanillin or tyrosol, among others, were present. This is the first time that HSCCC has been used to characterize the phenolic composition of Sherry wines.

Analytical Characterization and Sensory Analysis of Distillates of Different Varieties of Grapes Aged by an Accelerated Method.

The wine spirits used for the elaboration of Brandy de Jerez are mainly obtained from wines produced from the Airén type of grape, which comes from the vineyards located in the region of La Mancha (Central Spain). This entails a limitation when achieving a product classified as "protected geographic designation". For that purpose, it is necessary that the grape used for the wine spirit comes from the area and not from Castile la Mancha, as has happened until now. Due to this fact, it is necessary to search for a possible alternative grape variety which allows the produced brandy to be eligible for a "protected geographic designation". For that purpose, an accelerated ageing process has been implemented with a method previously optimized to distillates obtained from wines from different varieties of grapes (Airén, Colombard, Corredera, Doradilla, Garrido Fino, Jaén blanco, Moscatel de Alejandría, Palomino Fino, Ugni Blanc, and Zalema) grown in the Jerez Area. They were evaluated, both from the analytical and sensory points of view. The distillates made from Jaén Blanco and Zalema have properties that make them interesting for future development and incorporation into oenological practice.

Development and validation of UPLC for the determination of phenolic compounds and furanic derivatives in Brandy de Jerez.

Brandy and other aged distillates are a rich source of polyphenols. For brandies, contact with wood during ageing makes an important contribution to their polyphenols content. This paper describes the use of a previously devised ultra performance LC (UPLC) method to study the polyphenols content of Brandy de Jerez. UPLC is a new technique in LC offering several potential advantages, especially the reduction of time. Analyses of brandy performed by HPLC were repeated by UPLC. A special UPLC analytical column (Acquity UPLC BEH C18 column, 100 x 2.1 mm), with a particle size of 1.7 microm, forms part of this system. Using the UPLC system enabled the time needed for analysis to be reduced to one tenth of the time needed in the conventional HPLC system. In conclusion, the separation factor results of the UPLC were compared to those obtained using HPLC methods; this demonstrated that simple, high efficiency UPLC gradients are viable and advantageous substitutes for traditional analysis of polyphenols in brandy by HPLC. The method enabled 14 phenolic compounds to be identified and determined in 33 different commercial brandies, and this allowed them to be differentiated in function of quality.

Characterization and Differentiation of Spanish Vinegars from Jerez and Condado de Huelva Protected Designations of Origin.

Thirty one Jerez vinegar samples and 33 Huelva vinegar samples were analyzed for polyphenolic and volatile compound content in order to characterize them and attempt to differentiate them. Sixteen polyphenolic compounds were quantified by means of ultraperformance liquid chromatography method with diode array detection (UPLC-DAD), and 37 volatile compounds were studied by means of stir bar sorptive extraction-gas chromatography-mass spectrometry (SBSE-GC-MS). Spectrophotometric CIELab parameters were also measured for all the samples. The results obtained from the statistical multivariate treatment of the data evidenced a clear difference between vinegars from the two geographical indications with regard to their polyphenolic content, with Jerez vinegars exhibiting a greater phenolic content. Differentiation by the volatile compound content was, however, not so evident. Nevertheless, a considerable differentiation between the two groups of vinegars based on their volatile fraction was achieved. This may bring to light the grape varieties and geographical factors that have a clear influence on such differences.

High-Throughput Metabolomics Based on Direct Mass Spectrometry Analysis in Biomedical Research.

Metabolomics based on direct mass spectrometry analysis shows a great potential in biomedical research because of its high-throughput screening capability and wide metabolome coverage. This chapter contains detailed protocols to perform comprehensive metabolomic fingerprinting of multiple biological samples (serum, plasma, urine, brain, liver, spleen, thymus) by using complementary analytical platforms. The most important issues to be considered are discussed, including sample treatment, metabolomic analysis, raw data preprocessing, and data analysis.

Obesity induced alterations in redox homeostasis and oxidative stress are present from an early age.

OBJECTIVES: Oxidative stress and inflammation have been postulated as underlying mechanisms for the development of obesity-related insulin resistance. This association however, remains elusive especially in childhood. We sought to investigate this relation by measuring oxidative stress and antioxidant response biomarkers, before and during an oral glucose tolerance test (OGTT), in different biological samples from obese children. SUBJECTS: 24 children were recruited for the study, (18 obese and 6 controls). After OGTT, the obese group was subdivided in two, according to whether or not carbohydrate metabolic impairment (Ob.IR+, Ob.IR-; respectively) was found. Different biomarkers were analyzed after fasting (T = 0) and during an OGTT (T = 60 and 120 min). Lipoperoxides were measured in plasma, erythrocytes, and urine; while advanced glycation end products were determined in plasma, and redox status (GSH/GSSG ratio) in erythrocytes. RESULTS: We found marked differences in the characterization of the oxidative status in urine and erythrocytes, and in the dynamics of the antioxidant response during OGTT. Specifically, Ob.IR+ children show increased oxidative stress, deficient antioxidant response and a significant imbalance in redox status, in comparison to controls and Ob.IR- children. CONCLUSION: Obese children with insulin resistance show increased levels of oxidative stress biomarkers, and a stunted antioxidant response to an OGTT leading to increased oxidative stress after a single glucose load, as detected in erythrocytes, but not in plasma. We propose erythrocytes as sensors of early and acute changes in oxidative stress associated with insulin resistance in childhood obesity. This is a pilot study, performed with a limited sample size, so data should be interpreted with caution until reproduced.

An Overview on the Importance of Combining Complementary Analytical Platforms in Metabolomic Research.

The analytical bias introduced by most of the commonly used techniques in metabolomics considerably hinders the simultaneous detection of all metabolites present in complex biological samples. In order to solve this limitation, the combination of complementary approaches is emerging in recent years as the most suitable strategy in order to maximize metabolite coverage. This review article presents a general overview of the most important analytical techniques usually employed in metabolomics: nuclear magnetic resonance, mass spectrometry and hybrid approaches. Furthermore, we emphasize the potential of integrating various tools in the form of metabolomic multi-platforms in order to get a deeper metabolome characterization, for which a revision of the existing literature in this field is provided. This review is not intended to be exhaustive but, rather, to give a practical and concise guide to readers not familiar with analytical chemistry on the considerations to account for the proper selection of the technique to be used in a metabolomic experiment in biomedical research.

Noncontinuously binding loop-out primers for avoiding problematic DNA sequences in PCR and sanger sequencing.

We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.

Evolution of the colour, antioxidant activity and polyphenols in unusually aged Sherry wines.

The present paper reports the results of a study monitoring several key analytical parameters in a set of aged samples of vintages from 1999 back to 1935. The analysed parameters were: colour, antioxidant activity, low molecular weight phenolics, index of total polyphenols and SO2 content. It can be concluded that the antioxidant activity of the old wines studied is clearly related to the ageing time and to the polyphenols extracted from the wood during this period. The wines also showed differences in their chromatic characteristics according to the duration of their ageing in years. A principal component analysis confirmed that most of the studied variables (except hydroximethylfurfural and SO2) are strictly linked to ageing, which allowed a discrimination of 100% of the wines belonging to different decades, according to the results obtained by means of the application of linear discriminant analysis.

Concordance of butyrylcholinesterase phenotype with genotype: implications for biochemical reporting.

Butyrylcholinesterase (BChE) metabolizes the paralytic succinylcholine. Extended paralysis occurs in people with inherited BChE variants that may be identified by measuring BChE activity with and without the inhibitor dibucaine to calculate a dibucaine number (DN). Accurate phenotyping requires phenotype-specific BChE and DN reference intervals. We investigated the concordance between the biochemical BChE phenotype and the BCHE genotype to establish interpretive criteria for biochemical results. DNA was extracted from 45 serum specimens for which BChE activity and DN had been determined. The BCHE gene coding region was amplified and sequenced. Phenotype-genotype concordance and discordance occurred in 16 (36%) and 15 (33%) of specimens, respectively. A phenotype could not be assigned for 14 specimens (31%). An incorrectly assigned phenotype did not change the risk of prolonged paralysis or implied a slightly increased risk when there was none. Accurate BChE phenotyping is difficult using only enzyme activity and DN. The combination of biochemistry and BCHE genotype could improve the assessment of patient risk.

Oseltamivir-resistant influenza A 2009 H1N1 virus in immunocompromised patients.

BACKGROUND: First-line treatment of influenza A 2009 H1N1 relies on neuraminidase inhibitors such as oseltamivir. Resistance conferred by the H275Y neuraminidase gene mutation is concerning and likely to increase. OBJECTIVES: To characterize oseltamivir resistance in a hospital-based patient population. PATIENTS AND METHODS: All available respiratory specimens positive for influenza A by direct fluorescent antibody, RT-PCR, or culture from patients at the University of Utah 5/09-12/09 were collected. Specimens were confirmed as 2009 H1N1 by the Utah Department of Health. RT-PCR and pyrosequencing were used to test for the H275Y mutation (CDC protocol). PyroMark Q24 AQ software (Qiagen, Valencia, CA, USA) was used to allow for quantitative H275Y mutation analysis. Medical records of patients with resistant virus were reviewed. RESULTS: We tested 191 influenza A virus-positive samples from 187 unique patients. Fifty (27%) patients were hospitalized. Four patient specimens (2.1%) were found to carry the H275Y mutation. Three patients were hospitalized, representing 6% of inpatient samples tested. Three patients had undergone hematopoietic stem cell transplant in the past year. Two patients died. Their influenza viruses were confirmed to be oseltamivir-resistant at an independent reference laboratory and through the Center for Disease Control and Prevention (CDC). One patient reported no history of prior oseltamivir exposure. CONCLUSIONS: Widespread oseltamivir resistance among 2009 H1N1 remains a potential threat. Rapid techniques, such as pyrosequencing, which has the additional benefit of identifying mixed mutant populations of virus, may play a key role in identifying at-risk individuals and potentially unsuspected cases. Targeted surveillance of immunocompromised patients will be critical toward improving future influenza planning and therapy.