Optimization and validation of a UPLC-MS/MS assay for simultaneous quantification of 2,8-dihydroxyadenine, adenine, allopurinol, oxypurinol and febuxostat in human plasma.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare , hereditary disorder characterized by renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stone formation and chronic kidney disease (CKD). Treatment with a xanthine oxidoreductase inhibitor, allopurinol or febuxostat, reduces urinary DHA excretion and slows the progression of CKD. The method currently used for therapeutic monitoring of APRT deficiency lacks specificity and thus, a more reliable measurement technique is needed. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma was optimized and validated. Plasma samples were prepared with protein precipitation using acetonitrile followed by evaporation. The chemometric approach design of experiments was implemented to optimize gradient steepness, amount of organic solvent, flow rate, column temperature, cone voltage, desolvation temperature and desolvation flow rate. Experimental screening was conducted using fractional factorial design with addition of complementary experiments at the axial points for optimization of peak area, peak resolution and peak width. The assay was validated according to the US Food and Drug Administration guidelines for bioanalytical method validation over the concentration range of 50 to 5000 ng/mL for DHA, allopurinol and febuxostat, 100 to 5000 ng/mL for adenine and 50 to 12,000 ng/mL for oxypurinol, with r2 ≥ 0.99. The analytical assay achieved acceptable performance of accuracy (-10.8 to 8.3 %) and precision (CV < 15 %). DHA, adenine, allopurinol, oxypurinol and febuxostat were stable in plasma samples after five freeze-thaw cycles at -80 °C and after storage at -80 °C for 12 months. The assay was evaluated for quantification of the five analytes in clinical plasma samples from six APRT deficiency patients and proved to be both efficient and accurate. The proposed assay will be valuable for guiding pharmacotherapy and thereby contribute to improved and more personalized care for patients with APRT deficiency.

Metabolite Profile Characterization of Cyanobacterial Strains with Bioactivity on Lipid Metabolism Using In Vivo and In Vitro Approaches.

Cyanobacteria have demonstrated their therapeutic potential for many human diseases. In this work, cyanobacterial extracts were screened for lipid reducing activity in zebrafish larvae and in fatty-acid-overloaded human hepatocytes, as well as for glucose uptake in human hepatocytes and ucp1 mRNA induction in murine brown adipocytes. A total of 39 cyanobacteria strains were grown and their biomass fractionated, resulting in 117 chemical fractions. Reduction of neutral lipids in zebrafish larvae was observed for 12 fractions and in the human hepatocyte steatosis cell model for five fractions. The induction of ucp1 expression in murine brown adipocytes was observed in six fractions, resulting in a total of 23 bioactive non-toxic fractions. All extracts were analyzed by untargeted UPLC-Q-TOF-MS mass spectrometry followed by multivariate statistical analysis to prioritize bioactive strains. The metabolite profiling led to the identification of two markers with lipid reducing activity in zebrafish larvae. Putative compound identification using mass spectrometry databases identified them as phosphatidic acid and aromatic polyketides derivatives-two compound classes, which were previously associated with effects on metabolic disorders. In summary, we have identified cyanobacterial strains with promising lipid reducing activity, whose bioactive compounds needs to be identified in the future.

Performance Assessment of a 125 Human Plasma Peptide Mixture Stored at Room Temperature for Multiple Reaction Monitoring-Mass Spectrometry.

Synthetic peptides are a critical requirement for the development and application of targeted mass spectrometry (MS)-based assays for the quantitation of proteins from biological matrices. Transporting synthetic peptides on dry ice from one laboratory to another is costly and often difficult because of country-specific import and export regulations. Therefore, in this study, we assessed the impact of leaving a lyophilized mixture consisting of 125 peptides at room temperature for up to 20 days, and we assessed the effect on the quantitative performance of multiple reaction monitoring-MS (MRM-MS) assays. The findings suggest that there are no significant differences in the MRM-MS results for the time points assessed in this study (up to 20 days). All the calibration curves and quality control (QC) samples met the acceptance criteria for precision and accuracy (raw data are available via the public MS data repository PanoramaWeb, identifier: /MRM Proteomics/2020_BAK125_RT). The number of endogenous proteins quantifiable across five plasma samples was consistently between 87 and 99 out of 125 for all time points. Moreover, the coefficients of variation (CVs) calculated for the majority of peptide concentrations across all samples and time points were <5%. In addition, a lyophilized peptide mixture was transported from Canada to Iceland without dry ice. The results showed that there was no significant difference in the quantitative performance, with the determined concentrations of most proteins in the samples falling within 30% between the analyses performed on the same three plasma samples in Iceland and those in Canada. Overall, a comparison of the results obtained in Canada and in Iceland indicated that the peptides were stable under the conditions tested and also indicated that shipping lyophilized peptide mixtures without dry ice, but in the presence of sufficient desiccant material, could be a feasible option in cases where transport difficulties may arise or dry-ice sublimation may occur.

Genetic variability in the absorption of dietary sterols affects the risk of coronary artery disease.

AIMS: To explore whether variability in dietary cholesterol and phytosterol absorption impacts the risk of coronary artery disease (CAD) using as instruments sequence variants in the ABCG5/8 genes, key regulators of intestinal absorption of dietary sterols. METHODS AND RESULTS: We examined the effects of ABCG5/8 variants on non-high-density lipoprotein (non-HDL) cholesterol (N up to 610 532) and phytosterol levels (N = 3039) and the risk of CAD in Iceland, Denmark, and the UK Biobank (105 490 cases and 844 025 controls). We used genetic scores for non-HDL cholesterol to determine whether ABCG5/8 variants confer greater risk of CAD than predicted by their effect on non-HDL cholesterol. We identified nine rare ABCG5/8 coding variants with substantial impact on non-HDL cholesterol. Carriers have elevated phytosterol levels and are at increased risk of CAD. Consistent with impact on ABCG5/8 transporter function in hepatocytes, eight rare ABCG5/8 variants associate with gallstones. A genetic score of ABCG5/8 variants predicting 1 mmol/L increase in non-HDL cholesterol associates with two-fold increase in CAD risk [odds ratio (OR) = 2.01, 95% confidence interval (CI) 1.75-2.31, P = 9.8 × 10-23] compared with a 54% increase in CAD risk (OR = 1.54, 95% CI 1.49-1.59, P = 1.1 × 10-154) associated with a score of other non-HDL cholesterol variants predicting the same increase in non-HDL cholesterol (P for difference in effects = 2.4 × 10-4). CONCLUSIONS: Genetic variation in cholesterol absorption affects levels of circulating non-HDL cholesterol and risk of CAD. Our results indicate that both dietary cholesterol and phytosterols contribute directly to atherogenesis.

Untargeted metabolic profiling reveals geography as the strongest predictor of metabolic phenotypes of a cosmopolitan weed.

Plants produce a multitude of metabolites that contribute to their fitness and survival and play a role in local adaptation to environmental conditions. The effects of environmental variation are particularly well studied within the genus Plantago; however, previous studies have largely focused on targeting specific metabolites. Studies exploring metabolome-wide changes are lacking, and the effects of natural environmental variation and herbivory on the metabolomes of plants growing in situ remain unknown. An untargeted metabolomic approach using ultra-high-performance liquid chromatography-mass spectrometry, coupled with variation partitioning, general linear mixed modeling, and network analysis was used to detect differences in metabolic phenotypes of Plantago major in fifteen natural populations across Denmark. Geographic region, distance, habitat type, phenological stage, soil parameters, light levels, and leaf area were investigated for their relative contributions to explaining differences in foliar metabolomes. Herbivory effects were further investigated by comparing metabolomes from damaged and undamaged leaves from each plant. Geographic region explained the greatest number of significant metabolic differences. Soil pH had the second largest effect, followed by habitat and leaf area, while phenological stage had no effect. No evidence of the induction of metabolic features was found between leaves damaged by herbivores compared to undamaged leaves on the same plant. Differences in metabolic phenotypes explained by geographic factors are attributed to genotypic variation and/or unmeasured environmental factors that differ at the regional level in Denmark. A small number of specialized features in the metabolome may be involved in facilitating the success of a widespread species such as Plantago major into such wide range of environmental conditions, although overall resilience in the metabolome was found in response to environmental parameters tested. Untargeted metabolomic approaches have great potential to improve our understanding of how specialized plant metabolites respond to environmental change and assist in adaptation to local conditions.

DNA barcoding and LC-MS metabolite profiling of the lichen-forming genus Melanelia: Specimen identification and discrimination focusing on Icelandic taxa.

Taxa in the genus Melanelia (Parmeliaceae, Ascomycota) belong to a group of saxicolous lichens with brown to black foliose thalli, which have recently undergone extensive changes in circumscription. Taxa belonging to Parmeliaceae are prolific producers of bioactive compounds, which have also been traditionally used for chemotaxonomic purposes. However, the chemical diversity of the genus Melanelia and the use of chemical data for species discrimination in this genus are largely unexplored. In addition, identification based on morphological characters is challenging due to few taxonomically informative characters. Molecular identification methods, such as DNA barcoding, have rarely been applied to this genus. This study aimed to identify the Melanelia species from Iceland using DNA barcoding approach, and to explore their chemical diversity using chemical profiling. Chemometric tools were used to see if lichen metabolite profiles determined by LC-MS could be used for the identification of Icelandic Melanelia species. Barcoding using the fungal nuclear ribosomal internal transcribed spacer region (nrITS) successfully identified three Melalenlia species occurring in Iceland, together with Montanelia disjuncta (Basionym: Melanelia disjuncta). All species formed monophyletic clades in the neighbor-joining nrITS gene tree. However, high intraspecific genetic distance of M. stygia suggests the potential of unrecognized species lineages. Principal component analysis (PCA) of metabolite data gave a holistic overview showing that M. hepatizon and M. disjuncta were distinct from the rest, without the power to separate M. agnata and M. stygia due to their chemical similarity. Orthogonal partial least-squares to latent structures-discriminate analysis (OPLS-DA), however, successfully distinguished M. agnata and M. stygia by identifying statistically significant metabolites, which lead to class differentiation. This work has demonstrated the potential of DNA barcoding, chemical profiling and chemometrics in identification of Melanelia species.

Quantitative UPLC-MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency.

Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for quantification of urinary DHA. The UPLC-MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5min. Measurement of DHA in 24h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563ng/mL and 24h excretion of 816, 1327 and 1649mg, respectively. A rapid and robust UPLC-MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.

Severe osteoarthritis of the hand associates with common variants within the ALDH1A2 gene and with rare variants at 1p31.

Osteoarthritis is the most common form of arthritis and is a major cause of pain and disability in the elderly. To search for sequence variants that confer risk of osteoarthritis of the hand, we carried out a genome-wide association study (GWAS) in subjects with severe hand osteoarthritis, using variants identified through the whole-genome sequencing of 2,230 Icelanders. We found two significantly associated loci in the Icelandic discovery set: at 15q22 (frequency of 50.7%, odds ratio (OR) = 1.51, P = 3.99 × 10(-10)) in the ALDH1A2 gene and at 1p31 (frequency of 0.02%, OR = 50.6, P = 9.8 × 10(-10)). Among the carriers of the variant at 1p31 is a family with several members in whom the risk allele segregates with osteoarthritis. The variants within the ALDH1A2 gene were confirmed in replication sets from The Netherlands and the UK, yielding an overall association of OR = 1.46 and P = 1.1 × 10(-11) (rs3204689).