Small molecule biomarker discovery: Proposed workflow for LC-MS-based clinical research projects

Mass spectrometry focusing on small endogenous molecules has become an integral part of biomarker discovery in the pursuit of an in-depth understanding of the pathophysiology of various diseases, ultimately enabling the application of personalized medicine. While LC-MS methods allow researchers to gather vast amounts of data from hundreds or thousands of samples, the successful execution of a study as part of clinical research also requires knowledge transfer with clinicians, involvement of data scientists, and interactions with various stakeholders.The initial planning phase of a clinical research project involves specifying the scope and design, and engaging relevant experts from different fields. Enrolling subjects and designing trials rely largely on the overall objective of the study and epidemiological considerations, while proper pre-analytical sample handling has immediate implications on the quality of analytical data. Subsequent LC-MS measurements may be conducted in a targeted, semi-targeted, or non-targeted manner, resulting in datasets of varying size and accuracy. Data processing further enhances the quality of data and is a prerequisite for in-silico analysis. Nowadays, the evaluation of such complex datasets relies on a mix of classical statistics and machine learning applications, in combination with other tools, such as pathway analysis and gene set enrichment. Finally, results must be validated before biomarkers can be used as prognostic or diagnostic decision-making tools. Throughout the study, quality control measures should be employed to enhance the reliability of data and increase confidence in the results.The aim of this graphical review is to provide an overview of the steps to be taken when conducting an LC-MS-based clinical research project to search for small molecule biomarkers

The FKBP51 Inhibitor SAFit2 Restores the Pain-Relieving C16 Dihydroceramide after Nerve Injury

Neuropathic pain is a pathological pain state with a broad symptom scope that affects patients after nerve injuries, but it can also arise after infections or exposure to toxic substances. Current treatment possibilities are still limited because of the low efficacy and severe adverse effects of available therapeutics, highlighting an emerging need for novel analgesics and for a detailed understanding of the pathophysiological alterations in the onset and maintenance of neuropathic pain. Here, we show that the novel and highly specific FKBP51 inhibitor SAFit2 restores lipid signaling and metabolism in nervous tissue after nerve injury. More specifically, we identify that SAFit2 restores the levels of the C16 dihydroceramide, which significantly reduces the sensitization of the pain-mediating TRPV1 channel and subsequently the secretion of the pro-inflammatory neuropeptide CGRP in primary sensory neurons. Furthermore, we show that the C16 dihydroceramide is capable of reducing acute thermal hypersensitivity in a capsaicin mouse model. In conclusion, we report for the first time the C16 dihydroceramide as a novel and crucial lipid mediator in the context of neuropathic pain as it has analgesic properties, contributing to the pain-relieving properties of SAFit2

Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research

The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis

Implementation of lipidomics in clinical routine: Can fluoride/citrate blood sampling tubes improve preanalytical stability?

The impact of preanalytical sample handling on lipid stability has been assessed in human plasma using targeted LC-MS/MS quantification of endocannabinoids, sphingolipids and LPA, complemented by non-targeted lipidomics screening with LC-QTOFMS. The study involved incubation of whole blood and plasma from healthy volunteers at room temperature or in ice water for time periods ranging from 20 min to 24 h. The impact of two different anticoagulants, K3EDTA and sodium fluoride/citrate, on lipid stability was evaluated. It was found that the concentrations determined for several endogenous lipids vary when whole blood and plasma samples are processed at room temperature, whereas the concentrations of most lipids were stable for 4 h in ice water. Surprisingly, the detected amounts of endocannabinoids 1- and 2-arachidonoyl glycerol and arachidonoyl ethanolamide increased markedly by 60, 95, and 30% in K3EDTA whole blood after storage in ice water for only 20 min. When using sodium fluoride/citrate blood collection tubes, the stability of several lipids, including that of the endocannabinoids, was improved. Accordingly, it is absolutely necessary to keep the blood sampling and plasma processing time below 1 h to avoid ex-vivo formation of endocannabinoids. It is worth mentioning that baseline lipid levels differ when using K3EDTA or sodium fluoride/citrate blood sampling tubes, which emphasizes the importance of traceability of reported plasma concentrations to the used anticoagulant

Survival states as indicators of learning performance and biological stress in refugee children: a cross-sectional study with a comparison group

Background: Our goal was to accurately detect young children at risk for long-term psychiatric disturbances after potentially traumatic experiences in the course of relocation. In addition to detailed assessment of parent-rated parent and child symptomatology, we focused on disruptive behaviors in the education environment summarized as survival states, as these frequently lead to clinical referral. Methods: We screened 52 refugee children aged 3-7 (M = 5.14 years, SD = 1.17) for symptoms of Posttraumatic Stress Disorder (PTSD) with the Child and Adolescent Trauma Screening (CATS) in parent rating. The parents' mental health was assessed using the Refugee Health Screener (RHS-15). Furthermore, the child's educators were asked to evaluate the pathological survival states of the child and we made a general assessment of the children's symptoms with the Strengths and Difficulties Questionnaire (SDQ) rated by parents and educators. Children in the refugee sample completed a working memory learning task (Subtest Atlantis from the Kaufmann Assessment Battery for Children, KABC-II) and delivered saliva samples for testing of the cortisol level. Results: The parental rating of their child's PTSD symptoms was significantly related to their own mental well-being (r = .50, p < .001). Children with survival states in educator ratings exhibited weaker learning performance (F = 3.49, p < .05) and higher evening cortisol levels (U = 113, z = ? 1.7, p < .05, one-tailed). Conclusions: Survival states are promising indicators for children's learning performance and distress level complementary to parent rating of child PTSD, which is highly intercorrelated with the parents' own symptom load. Trial registration number: DRKS00021150 on DRKS Date of registration: 04.08.2020 retrospectively registere

Survival states as indicators of learning performance and biological stress in refugee children: a cross-sectional study with a comparison group

Abstract Background Our goal was to accurately detect young children at risk for long-term psychiatric disturbances after potentially traumatic experiences in the course of relocation. In addition to detailed assessment of parent-rated parent and child symptomatology, we focused on disruptive behaviors in the education environment summarized as survival states, as these frequently lead to clinical referral. Methods We screened 52 refugee children aged 3–7 (M = 5.14 years, SD = 1.17) for symptoms of Posttraumatic Stress Disorder (PTSD) with the Child and Adolescent Trauma Screening (CATS) in parent rating. The parents’ mental health was assessed using the Refugee Health Screener (RHS-15). Furthermore, the child’s educators were asked to evaluate the pathological survival states of the child and we made a general assessment of the children’s symptoms with the Strengths and Difficulties Questionnaire (SDQ) rated by parents and educators. Children in the refugee sample completed a working memory learning task (Subtest Atlantis from the Kaufmann Assessment Battery for Children, KABC-II) and delivered saliva samples for testing of the cortisol level. Results The parental rating of their child’s PTSD symptoms was significantly related to their own mental well-being (r = .50, p < .001). Children with survival states in educator ratings exhibited weaker learning performance (F = 3.49, p < .05) and higher evening cortisol levels (U = 113, z = − 1.7, p < .05, one-tailed). Conclusions Survival states are promising indicators for children’s learning performance and distress level complementary to parent rating of child PTSD, which is highly intercorrelated with the parents’ own symptom load. Trial registration Trial registration number: DRKS00021150 on DRKS Date of registration: 04.08.2020 retrospectively registere

Non-verbal cognitive development, learning, and symptoms of PTSD in 3- to 6-year-old refugee children

As IQ tests are commonly used as key assessment method, we address the question whether our commonly used standardized IQ tests are appropriate for children from families of diverse cultures and different educational levels in a refugee population. We examined 109 refugee children aged 3-7 years (M = 5.10 years, SD = 1.25) with the 'Kaufman Assessment Battery for Children ' (KABC-II; Kaufmann &amp;amp; Kaufmann, 2015) on a language-free scale (Scale of Intellectual Functioning, SIF) and learning performance (subtest Atlantis). With a non-verbal IQ of 81.5 (SD = 18.01), the population mean of the refugee children is more than one standard deviation lower than the mean of the German norm population. Standardized scores follow the normal distribution and are not correlated to any of the assessed markers of adversity (flight duration, time spent in Germany, child PTSD in parent rating, parental symptom load, and parental education level).Conclusion: The interpretation of IQ test results for refugee children should be done cautiously as results may underestimate their cognitive capacity. Environmental factors, such as high illiteracy among parents in this study, the lack of institutional education of children and high lifetime stress, may explain our findings.Trial registration: DRKS00021150. What is Known: • There is a high pervasiveness for the use of standardized IQ tests in the German health and education system to determine eligibility for special education and social services. What is New: • Refugee children score significantly lower than German children in a language-free IQ test. As results are normally distributed and not correlated to any of the assessed markers of adversity, the low scores in the refugee group might be due to missing formal education

Targeted lipidomics reveal derangement of ceramides in major depression and bipolar disorder

Changes of sphingolipid metabolism were suggested to contribute to the patho-etiology of major depression (MD) and bipolar disorder (BD). In a pilot study we assessed if lipid allostasis manifested in pathological plasma concentrations of bioactive lipids i.e. endocannabinoids, sphingolipids, ceramides, and lysophosphatidic acids. Methods Targeted and untargeted lipidomic analyses were performed according to GLP guidelines in 67 patients with unipolar or bipolar disorders (20–67 years, 36 male, 31 female) and 405 healthy controls (18–79 years, 142 m, 263 f), who were matched according to gender, age and body mass index. Multivariate analyses were used to identify major components, which accounted for the variance between groups and were able to predict group membership. Results Differences between MD and BP patients versus controls mainly originated from ceramides and their hexosyl-metabolites (C16Cer, C18Cer, C20Cer, C22Cer, C24Cer and C24:1Cer; C24:1GluCer, C24LacCer), which were strongly increased, particularly in male patients. Ceramide levels were neither associated with the current episode, nor with the therapeutic improvement of the Montgomery Åsberg Depression Rating Scale (MARDS). However, long-chain ceramides were linearly associated with age, stronger in patients than controls, and with high plasma levels of diacyl- and triacylglycerols. Patients receiving antidepressants had higher ceramide levels than patients not taking these drugs. There was no such association with lithium or antipsychotics except for olanzapine. Conclusion Our data suggest that high plasma ceramides in patients with major depression and bipolar disorder are indicative of a high metabolic burden, likely aggravated by certain medications