Therese Koal

Targeted Metabolomics for Biomarker Discovery

Metabolomics is a truly interdisciplinary field of science, which combines analytical chemistry, platform technology, mass spectrometry, and NMR spectroscopy with sophisticated data analysis. Applied to biomarker discovery, it includes aspects of pathobiochemistry, systems biology/medicine, and molecular diagnostics and requires bioinformatics and multivariate statistics. While successfully established in the screening of inborn errors in neonates, metabolomics is now widely used in the characterization and diagnostic research of an ever increasing number of diseases. In this Review we highlight important technical prerequisites as well as recent developments in metabolomics and metabolomics data analysis with special emphasis on their utility in biomarker identification and qualification, as well as targeted metabolomics by employing high-throughput mass spectrometry.

Standardized LC-MS/MS based steroid hormone profile-analysis.

In order to overcome many limitations of immunoassays, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC-MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01-32 ng/mL, ULOQ 5-8000 ng/mL), linear correlation coefficient of calibration (R(2)>0.9966), intra- and inter-day precision (intra-day 1.1-8.8%, inter-day 5.2-14.8% and 8.2-18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3-115.5% and 109.3-128.2% for 11-deoxycorticosterone, inter-day 91.4-117.2% and 102.3-137.1% for 11-deoxycorticosterone), analytical total error (3.6-17.8%), proficiency test accuracy (85.4-113.4%), recovery (68-99%), and metabolite stability (freeze/thaw stability 95.5-108.1%, short term stability 86.9-107.2%). Inter-assay comparison with a routine reference HPLC-MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC-MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC-MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.

Metabolomic Analyses of Plasma Reveals New Insights into Asphyxia and Resuscitation in Pigs

Background Currently, a limited range of biochemical tests for hypoxia are in clinical use. Early diagnostic and functional biomarkers that mirror cellular metabolism and recovery during resuscitation are lacking. We hypothesized that the quantification of metabolites after hypoxia and resuscitation would enable the detection of markers of hypoxia as well as markers enabling the monitoring and evaluation of resuscitation strategies. Methods and Findings Hypoxemia of different durations was induced in newborn piglets before randomization for resuscitation with 21% or 100% oxygen for 15 min or prolonged hyperoxia. Metabolites were measured in plasma taken before and after hypoxia as well as after resuscitation. Lactate, pH and base deficit did not correlate with the duration of hypoxia. In contrast to these, we detected the ratios of alanine to branched chained amino acids (Ala/BCAA; R2.adj = 0.58, q-value<0.001) and of glycine to BCAA (Gly/BCAA; R2.adj = 0.45, q-value<0.005), which were highly correlated with the duration of hypoxia. Combinations of metabolites and ratios increased the correlation to R2adjust = 0.92. Reoxygenation with 100% oxygen delayed cellular metabolic recovery. Reoxygenation with different concentrations of oxygen reduced lactate levels to a similar extent. In contrast, metabolites of the Krebs cycle (which is directly linked to mitochondrial function) including alpha keto-glutarate, succinate and fumarate were significantly reduced at different rates depending on the resuscitation, showing a delay in recovery in the 100% reoxygenation groups. Additional metabolites showing different responses to reoxygenation include oxysterols and acylcarnitines (n = 8–11, q<0.001). Conclusions This study provides a novel strategy and set of biomarkers. It provides biochemical in vivo data that resuscitation with 100% oxygen delays cellular recovery. In addition, the oxysterol increase raises concerns about the safety of 100% O2 resuscitation. Our biomarkers can be used in a broad clinical setting for evaluation or the prediction of damage in conditions associated with low tissue oxygenation in both infancy and adulthood. These findings have to be validated in human trials.

Rapid sample preparation and simultaneous quantitation of prostaglandins and lipoxygenase derived fatty acid metabolites by liquid chromatography-mass spectrometry from small sample volumes

Abstract Background: Fatty acid metabolites play a key role in numerous physiological and pathological processes. A rapid liquid chromatography-mass spectrometry assay for the simultaneous determination of prostanoids, isoprostane and lipoxygenase (LOX) derived fatty acid metabolites in a small biological sample of only 20 μL was developed. Methods: Human plasma samples were applied to a filter spot, extracted without prior derivatization and analyzed within 13 min. Detection of metabolites was performed on a triple quadrupole mass spectrometer in negative multiple-reaction monitoring detection mode. Application of this assay to various biological matrices was performed. Results: The validated assay was linear over the concentration range of 5–500 nmol/L for prostanoids and isoprostane, 50–5000 nmol/L for LOX-derived metabolites and 400–40,000 nmol/L for fatty acids. Limits of quantitation were 0.4–233 nmol/L, depending on the metabolite. Plasma samples from diabetic patients and controls showed significant increases in (±)9-HODE and 15(S)-HETE with p-values of 0.019 and 0.024, respectively. Conclusions: The small amount of 20 μL sample volume used in this assay and the demonstrated application to various sample types makes it an ideal routine analysis method for fatty acid metabolites. The resulting values for LOX-derived metabolites in diabetes mellitus type 2 samples support earlier findings about the role of lipid oxidation products in diabetes. Clin Chem Lab Med 2008;46:1589–97.

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma.

Abstract Background: In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma® system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. Methods: The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-μL sample volume, which was only diluted with 200μL of H2O (containing 500ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. Results: The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6min, subsequent analyses 3.3min between injections) and has been validated in terms of the limit of detection (LOD, 2–70ng/mL), lower limit of quantification (LLOQ, 78–156ng/mL), linearity (R2, 0.9971–0.9989), linear concentration range (from LLOQ to 10,000ng/mL), intra- and inter-day precision (<13.5% at LLOQ, <7.5% at high concentrations), proficiency testing accuracy (78–127%), laboratory internal accuracy (86–113%), recovery (60–110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. Conclusion: Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.

Isotope correction of mass spectrometry profiles

Isotope correction of a profile is an important step in the analysis of mass spectrometry derived data. The problem is mathematically formulated as a system of linear equations which is general enough to include previous correction methods. For the solution of these equations when applied to the whole profile an efficient algorithm is developed. In experimental tests the resulting algorithm corrected the profile fast and successfully.

Complexity and pitfalls of mass spectrometry-based targeted metabolomics in brain research.

Current quantitative metabolomic research in brain tissue is challenged by several analytical issues. To compare data of metabolite pattern, ratios of individual metabolite concentrations and composed classifiers characterizing a distinct state, standardized workup conditions, and extraction medium are crucial. Differences in physicochemical properties of individual compounds and compound classes such as polarity determine extraction yields and, thus, ratios of compounds with varying properties. Also, variations in suppressive effects related to coextracted matrix components affect standards or references and their concentration-dependent responses.The selection of a common tissue extraction protocol is an ill-posed problem because it can be regarded as a multiple objective decision depending on factors such as sample handling practicability, measurement precision, control of matrix effects, and relevance of the chemical assay. This study systematically evaluates the impact of extraction solvents and the impact of the complex brain tissue on measured metabolite levels, taking into account ionization efficiency as well as challenges encountered in the trace-level quantification of the analytes in brain matrices. In comparison with previous studies that relied on nontargeted platforms, consequently emphasizing the global behavior of the metabolomic fingerprint, here we focus on several series of metabolites spanning over extensive polarity, concentration, and molecular mass ranges.

Metabolic network failures in Alzheimer's disease—A biochemical road map

The Alzheimers Disease Research Summits of 2012 and 2015 incorporated experts from academia, industry, and nonprofit organizations to develop new research directions to transform our understanding of Alzheimers disease (AD) and propel the development of critically needed therapies. In response to their recommendations, big data at multiple levels are being generated and integrated to study network failures in disease. We used metabolomics as a global biochemical approach to identify peripheral metabolic changes in AD patients and correlate them to cerebrospinal fluid pathology markers, imaging features, and cognitive performance.

The ratio of phosphatidylcholines to lysophosphatidylcholines in plasma differentiates healthy controls from patients with Alzheimer's disease and mild cognitive impairment.

Metabolomic processes have been identified as being strongly linked to the development of Alzheimers disease (AD). Thus, lipid metabolites appear to be highly useful as diagnostic substrates for the diagnosis of AD and mild cognitive impairment (MCI) in plasma.

Everolimus/cyclosporine interactions on bile flow and biliary excretion of bile salts and cholesterol in rats.

As a possible explanation for everolimus/cyclosporine-induced hypercholesterolemia seen in transplant recipients, we investigated the interactions of the immunosuppressants everolimus and cyclosporine on bile flow and biliary excretion of bile salts and cholesterol in a subchronic bile fistula model in rats because biliary excretion is a main elimination route of cholesterol. After 2 weeks of daily treatment, everolimus (1 mg/kg i.p.) and cyclosporine (5 mg/kg i.p) decreased bile flow (−45 and −36%) and biliary excretion of bile salts (−34 and −54%) and cholesterol (−25 and −39%) and increased serum concentrations of cholesterol (+40 and +17%) and triglycerides (+220 and +110%). Bile salt serum concentration was elevated only by cyclosporine (+100%), and not by everolimus. Everolimus/cyclosporine slightly enforced the cyclosporine-induced hyperlipidemia but not reduction of bile parameters, while the cyclosporine-induced increase in bile salts in serum was totally prevented. From these results we conclude that bile salt synthesis could be impaired by everolimus, which could be one reason for everolimus-induced hypercholesterolemia.