Lipidomic changes occurring in platelets during extended cold storage.

OBJECTIVES: Cold storage is being implemented as an alternative to conventional room-temperature storage for extending the shelf-life of platelet components beyond 5-7 days. The aim of this study was to characterise the lipid profile of platelets stored under standard room-temperature or cold (refrigerated) conditions. METHODS: Matched apheresis derived platelet components in 60% PAS-E/40% plasma (n = 8) were stored at room-temperature (20-24°C with agitation) or in the cold (2-6°C without agitation). Platelets were sampled on day 1, 5 and 14. The lipidome was assessed by ultra-pressure liquid chromatography ion mobility quadrupole time of flight mass spectrometry (UPLC IMS QToF). Changes in bioactive lipid mediators were measured by ELISA. RESULTS: The total phospholipid and sphingolipid content of the platelets and supernatant were 44 544 ± 2915 µg/mL and 38 990 ± 10 880 µg/mL, respectively, and was similar over 14 days, regardless of storage temperature. The proportion of the procoagulant lipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), increased by 2.7% and 12.2%, respectively, during extended cold storage. Cold storage for 14 days increased sphingomyelin (SM) by 4.1% and decreased ceramide by 1.6% compared to day 1. Further, lysophosphatidylcholine (LPC) species remained unchanged during cold storage for 14 days. The concentration of 12- and 15-hydroxyeicosatetraenoic acid (HETE) were lower in the supernatant of cold-stored platelets than room-temperature controls stored for 14 days. CONCLUSION: The lipid profile of platelets was relatively unchanged during storage for 5 days, regardless of temperature. However, during extended cold storage (14 days) the proportion of the procoagulant lipids, PS and PE, increased, while LPC and bioactive lipids were stable.

Automated Structural Activity Screening of ß-Diketonate Assemblies with High-Throughput Ion Mobility-Mass Spectrometry.

Embracing complexity in design, metallo-supramolecular self-assembly presents an opportunity for fabricating materials of economic significance. The array of accessible supramolecules is alluring, along with favourable energy requirements. Implementation is hampered by an inability to efficiently characterise complex mixtures. The stoichiometry, size, shape, guest binding properties and reactivity of individual components and combinations thereof are inherently challenging to resolve. A large combinatorial library of four transition metals (Fe, Cu, Ni and Zn), and six ß-diketonate ligands at different molar ratios and pH was robotically prepared and directly analysed over multiple timepoints with electrospray ionisation travelling wave ion mobility-mass spectrometry. The dataset was parsed for self-assembling activity without first attempting to structurally assign individual species. Self-assembling systems were readily categorised without manual data-handling, allowing efficient screening of self-assembly activity. This workflow clarifies solution phase supramolecular assembly processes without manual, bottom-up processing. The complex behaviour of the self-assembling systems was reduced to simpler qualities, which could be automatically processed.

"Simultaneous targeted and non-targeted analysis of per- and polyfluoroalkyl substances in environmental samples by liquid chromatography-ion mobility-quadrupole time of flight-mass spectrometry and mass defect analysis".

Per- and polyfluoroalkyl substances (PFAS) represent a large group of synthetic organic compounds which exhibit unique properties and have been extensively used for consumer and industrial products, resulting in a widespread presence in the environment. Regulation requiring PFAS monitoring has been implemented worldwide due to their potential health and eco-toxicological effects. Targeted methods are commonly used to monitor between twenty to forty PFAS compounds, representing only a small fraction of the number of compounds that may be present. Consequently, there is an increasing interest in complementary non-targeted methods to screen and identify unknown PFAS compounds with the aim to improve knowledge and to generate more accurate models regarding their environmental mobility and persistence. This work details the development of a method that simultaneously provided targeted and non-targeted PFAS analysis. Ultra-high performance liquid chromatography (UHPLC) was coupled to ion mobility-quadrupole time of flight-mass spectrometry (IMS-QTOF-MS) and used to quantify known and screen unknown PFAS in environmental samples collected within the greater Sydney basin (Australia). The method was validated for the quantification of 14 sulfonate-based PFAS, and a non-targeted data analysis workflow was developed using a combination of mass defect analysis with common fragment and neutral loss filtering to identify fluorine-containing species. The optimised method was applied to the environmental samples and enabled the determination of 3-7 compounds from the targeted list and the detection of a further 56-107 untargeted PFAS. This simultaneous analysis reduces the complexity of multiple analyses, and allows for greater interrogation of the full PFAS load in environmental samples.

Impact of CYP2D6 genotype on amitriptyline efficacy for the treatment of diabetic peripheral neuropathy: a pilot study.

AIM: Therapy with low-dose amitriptyline is commonly used to treat painful diabetic peripheral neuropathy. There is a knowledge gap, however, regarding the role of variable CYP2D6-mediated drug metabolism and side effects (SEs). We aimed to generate pilot data to demonstrate that SEs are more frequent in patients with variant CYP2D6 alleles. METHOD: To that end, 31 randomly recruited participants were treated with low-dose amitriptyline for painful diabetic peripheral neuropathy and their CYP2D6 gene sequenced. RESULTS: Patients with predicted normal or ultra-rapid metabolizer phenotypes presented with less SEs compared with individuals with decreased CYP2D6 activity. CONCLUSION: Hence, CYP2D6 genotype contributes to treatment outcome and may be useful for guiding drug therapy. Future investigations in a larger patient population are planned to support these preliminary findings.

Pharmacogenomics and Global Precision Medicine in the Context of Adverse Drug Reactions: Top 10 Opportunities and Challenges for the Next Decade.

In a move indicative of the enthusiastic support of precision medicine, the U.S. President Barack Obama announced the Precision Medicine Initiative in January 2015. The global precision medicine ecosystem is, thus, receiving generous support from the United States ($215 million), and numerous other governments have followed suit. In the context of precision medicine, drug treatment and prediction of its outcomes have been important for nearly six decades in the field of pharmacogenomics. The field offers an elegant solution for minimizing the effects and occurrence of adverse drug reactions (ADRs). The Clinical Pharmacogenetics Implementation Consortium (CPIC) plays an important role in this context, and it aims at specifically guiding the translation of clinically relevant and evidence-based pharmacogenomics research. In this forward-looking analysis, we make particular reference to several of the CPIC guidelines and their role in guiding the treatment of highly relevant diseases, namely cardiovascular disease, major depressive disorder, cancer, and human immunodeficiency virus, with a view to predicting and managing ADRs. In addition, we provide a list of the top 10 crosscutting opportunities and challenges facing the fields of precision medicine and pharmacogenomics, which have broad applicability independent of the drug class involved. Many of these opportunities and challenges pertain to infrastructure, study design, policy, and science culture in the early 21st century. Ultimately, rational pharmacogenomics study design and the acquisition of comprehensive phenotypic data that proportionately match the genomics data should be an imperative as we move forward toward global precision medicine.

Allelic variants of the Melanocortin 4 receptor (MC4R) gene in a South African study group.

Obesity is a global epidemic that results in significant morbidity and mortality. Mutations in the melanocortin 4 receptor (MC4R) gene, which codes for a G-protein-coupled receptor responsible for postprandial satiety signaling, have been associated with monogenic obesity. The prevalence of obesity is on the increase in South Africa, and it is hypothesized that mutations in MC4R are a contributing factor. The aim of this study was to perform a retrospective assessment of the relationship between allelic variants of MC4R and BMI in a South African study cohort. DNA was isolated from a demographically representative cohort of 297 individuals and the entire MC4R gene sequenced by Sanger sequencing. Eight previously reported MC4R variants were identified in 42 of the 297 (14.1%) study participants. The most frequently observed MC4R alleles were V103I (4.0%), I170V (1.5%), and I198I (1.2%), while the remaining five variants together constituted 1.18%. Five compound heterozygotes were also detected. Although MC4R variants were rare, the majority of variation was observed in individuals of Black African ancestry. No statistically significant associations with BMI were reported. Given that lifestyle interventions have limited success in decreasing obesity, there is an urgent need to perform large-scale population studies to further elucidate the molecular underpinnings of this disease.

Evaluation of predictive CYP2C19 genotyping assays relative to measured phenotype in a South African cohort.

AIM: To align predicted and measured CYP2C19 phenotype in a South African cohort. MATERIALS & METHODS: Genotyping of CYP2C19*2, *3, *9, *15, *17, *27 and *28 was performed using PCR-RFLP, and an activity score (AS) system was used to predict phenotype. True phenotype was measured using plasma concentrations of omeprazole and its metabolite 5'-hydroxyomperazole. RESULTS: Partial genotype-phenotype discrepancies were reported, and an adapted AS system was developed, which showed a marked improvement in phenotype prediction. Results highlight the need for a more comprehensive CYP2C19 genotyping approach to improve prediction of omeprazole metabolism. CONCLUSION: Evidence for the utility of a CYP2C19 AS system is provided, for which the accuracy can be further improved by means of comprehensive genotyping and substrate-specific modification.

Risperidone-associated adverse drug reactions and CYP2D6 polymorphisms in a South African cohort.

BACKGROUND: Contradictory information exists regarding the influence of CYP2D6 polymorphisms on adverse drug reactions (ADRs) (extrapyramidal symptoms (EPS) and weight gain) related to risperidone treatment. This prompted us to evaluate the influence of CYP2D6 genetic variation in a cohort of South African patients who presented with marked movement disorders and/or weight gain while on risperidone treatment. METHODS: Patients who were experiencing marked risperidone ADRs were recruited from Weskoppies Public Psychiatric Hospital. As poor or intermediate metabolism was expected, comprehensive CYP2D6 sequence variations were evaluated using XL-PCR + Sequencing. RESULTS: No statistically significant association was found between CYP2D6 poor metabolism and risperidone ADRs. An inverse relationship between EPS and weight gain was however identified. A novel CYP2D6 allele was identified which is unlikely to affect metabolism based on in silico evaluation. CONCLUSION: CYP2D6 variation appeared not to be a good pharmacogenetic marker for predicting risperidone-related ADRs in this naturalistic South African cohort. Evaluation of a larger cohort would be needed to confirm these observations, including an examination of the role of potential intermediaries between the hypothesised genetic and clinical phenotypes.

Introduction of the AmpliChip CYP450 Test to a South African cohort: a platform comparative prospective cohort study.

BACKGROUND: Adverse drug reactions and lack of therapeutic efficacy associated with currently prescribed pharmacotherapeutics may be attributed, in part, to inter-individual variability in drug metabolism. Studies on the pharmacogenetics of Cytochrome P450 (CYP) enzymes offer insight into this variability. The objective of this study was to compare the AmpliChip CYP450 Test® (AmpliChip) to alternative genotyping platforms for phenotype prediction of CYP2C19 and CYP2D6 in a representative cohort of the South African population. METHODS: AmpliChip was used to screen for thirty-three CYP2D6 and three CYP2C19 alleles in two different cohorts. As a comparison cohort 2 was then genotyped using a CYP2D6 specific long range PCR with sequencing (CYP2D6 XL-PCR + Sequencing) platform and a PCR-RFLP platform for seven CYP2C19 alleles. RESULTS: Even though there was a low success rate for the AmpliChip, allele frequencies for both CYP2D6 and CYP2C19 were very similar between the two different cohorts. The CYP2D6 XL-PCR + Sequencing platform detected CYP2D6*5 more reliably and could correctly distinguish between CYP2D6*2 and *41 in the Black African individuals. Alleles not covered by the AmpliChip were identified and four novel CYP2D6 alleles were also detected. CYP2C19 PCR-RFLP identified CYP2C19*9,*15, *17 and *27 in the Black African individuals, with *2, *17 and *27 being relatively frequent in the cohort. Eliminating mismatches and identifying additional alleles will contribute to improving phenotype prediction for both enzymes. Phenotype prediction differed between platforms for both genes. CONCLUSION: Comprehensive genotyping of CYP2D6 and CYP2C19 with the platforms used in this study, would be more appropriate than AmpliChip for phenotypic prediction in the South African population. Pharmacogenetically important novel alleles may remain undiscovered when using assays that are designed according to Caucasian specific variation, unless alternate strategies are utilised.

Cardiovascular pharmacogenetics.

Human genetic variation in the form of single nucleotide polymorphisms as well as more complex structural variations such as insertions, deletions and copy number variants, is partially responsible for the clinical variation seen in response to pharmacotherapeutic drugs. This affects the likelihood of experiencing adverse drug reactions and also of achieving therapeutic success. In this paper, we review key studies in cardiovascular pharmacogenetics that reveal genetic variations underlying the outcomes of drug treatment in cardiovascular disease. Examples of genetic associations with drug efficacy and toxicity are described, including the roles of genetic variability in pharmacokinetics (e.g. drug metabolizing enzymes) and pharmacodynamics (e.g. drug targets). These findings have functional implications that could lead to the development of genetic tests aimed at minimizing drug toxicity and optimizing drug efficacy in cardiovascular medicine.

Quantitative plasma analysis using automated online solid-phase extraction with column switching LC-MS/MS for characterising cytochrome P450 2D6 and 2C19 metabolism.

In this study an easy and efficient assay for simultaneous quantitation of plasma concentrations of probe drugs and their metabolically relevant metabolites for the phenotypic analysis of cytochrome P450 2D6 and 2C19, respectively, has been established. This sensitive method makes use of a simple initial sample preparation, followed by a 6-min automated analysis that includes online solid-phase extraction (SPE), column switching and tandem mass spectrometry. Validation over a concentration range of 1.3-2500 ng/mL for dextromethorphan, omeprazole, dextrorphan and 5'-hydroxyomeprazole was performed with LOQ between 215 and 1145 pg/mL. Intra- and inter-day precision and accuracy over the calibration ranges were within 15% for all analytes with recoveries of greater than 85%. Advantages are small sample volumes required, a robust, sensitive and highly selective method suitable for pre-prescription metabolic screening. This method could compliment or offer an alternative to DNA mutation analysis for determining appropriate dosage regimens for personalised medicine.