Method Development for Omics Analyses using Schirmer Strips.

PURPOSE: The aim of this article was to investigate whether Schirmer strips gathered during clinical dry eye examinations can be prepared for omics analyses in a standardized way, to adjust for variations in tear volume and enable two separate omics analyses from the same sample. In addition, the intention was to investigate whether fluorescein dye instillation in the eyes gave bias effects on metabolomic analysis. METHODS: Twelve samples from six individuals, with normal or reduced tear production, were collected. Half of the samples were harvested after instillation of fluorescein in the eye. Each strip was divided in half along the length and prepared with a new method for extracting tear content from the Schirmer strip. The new method was established to compensate for different dilutions of metabolites in varying Schirmer strip wetting levels when using identical extraction volume for all samples. Metabolomic data were compared in samples with and without fluorescein dye and Schirmer strips ranging from 1 to 35 mm wetting levels using a global LC-MS method. RESULTS: All samples were successfully analyzed with an average of ∼350 relevant features detected per sample after using both positive and negative electrospray ionization mode, despite low tear volumes in some samples and that only one half of the Schirmer strips were used. Principal component analysis plots and heatmaps revealed no bias effects of fluorescein dye presence or different Schirmer strip values when using the proposed method. CONCLUSION: A high number of relevant metabolomic features can be extracted from longitudinally cut halves of Schirmer strips, which may enable analyses with more than one omics modality from the same sample. With the pre-analytical method described, Schirmer strips can be used for metabolomic analyses even in cases of very low or high tear volume with or without fluorescence.

Ex Vivo Pig Eye Model for Studying Diffusion of Acylcarnitines with Different Physicochemical Properties.

Purpose: To explore the diffusion capacities between the anterior and vitreous chambers in a novel ex vivo pig eye model using a mix of stable isotope-labeled acylcarnitines representing metabolites with different physical and chemical properties, and analysis using mass spectrometry (MS). Methods: Enucleated pig eyes were injected in the anterior or vitreous chamber of the eye with a stable isotope-labeled acylcarnitine mix (free carnitine, C2, C3, C4, C8, C12, and C16-having an increasing size and hydrophobicity in that order). Samples were collected from each chamber at 3, 6, and 24 h postincubation for analysis using MS. Results: After injection into the anterior chamber, the concentration of all acylcarnitines increased in the vitreous chamber over the observation period. After injection in the vitreous chamber, the acylcarnitines diffused to the anterior chamber with the highest concentration observed at 3 h postinjection, followed by a decrease in concentration possibly due to an elimination from the anterior chamber despite continued diffusion from the vitreous chamber. C16, the most hydrophobic and longest chain molecule, showed slower diffusion in both experimental settings. Conclusion: We hereby show a distinct diffusion pattern of molecules with different molecular size and hydrophobicity within and between the anterior and vitreous chamber. This model can be useful for optimizing choices and design of therapeutic molecules with higher retaining or depot properties into the two chambers of the eye for future intravitreal, intracameral, and topical treatment purposes.

Global Metabolomics Discovers Two Novel Biomarkers in Pyridoxine-Dependent Epilepsy Caused by ALDH7A1 Deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive developmental and epileptic encephalopathy caused by pathogenic variants in the ALDH7A1 gene (PDE-ALDH7A1), which mainly has its onset in neonates and infants. Early diagnosis and treatment are crucial to prevent severe neurological sequelae or death. Sensitive, specific, and stable biomarkers for diagnostic evaluations and follow-up examinations are essential to optimize outcomes. However, most of the known biomarkers for PDE lack these criteria. Additionally, there is little discussion regarding the interdependence of biomarkers in the PDE-ALDH7A1 metabolite profile. Therefore, the aim of this study was to understand the underlying mechanisms in PDE-ALDH7A1 and to discover new biomarkers in the plasma of patients using global metabolomics. Plasma samples from 9 patients with genetically confirmed PDE-ALDH7A1 and 22 carefully selected control individuals were analyzed by ultra high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Two novel and reliable pyridoxine-independent diagnostic markers, 6-hydroxy-2-aminocaproic acid (HACA) and an isomer of C9H11NO4, were identified. Furthermore, a possible reaction mechanism is proposed for HACA. This study demonstrates the capability of global metabolomics in disease screening to detect established and novel biomarkers.

Saliva Metabolomics in Dry Mouth Patients with Head and Neck Cancer or Sjögren's Syndrome.

The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.

A pioneer study on human 3-nitropropionic acid intoxication: Contributions from metabolomics.

The neurotoxin 3-nitropropionic acid (3-NPA) is an inhibitor of succinate dehydrogenase, an enzyme participating both in the citric acid cycle and the mitochondrial respiratory chain. In human intoxications, it produces symptoms such as vomiting and stomach ache in mild cases, and dystonia, coma, and sometimes death in severe cases. We report the results from a liquid chromatography-Orbitrap mass spectrometry metabolomics study mapping the metabolic impacts of 3-NPA intoxication in plasma, urine, and cerebrospinal fluid (CSF) samples of a Norwegian boy initially suspected to suffer from a mitochondrial disease. In addition to the identification of 3-NPA, our findings included a large number of annotated/identified altered metabolites (80, 160, and 62 in plasma, urine, and CSF samples, respectively) belonging to different compound classes, for example, amino acids, fatty acids, and purines and pyrimidines. Our findings indicated protective mechanisms to attenuate the toxic effects of 3-NPA (e.g., decreased oleamide), occurrence of increased oxidative stress in the patient (such as increased free fatty acids and hypoxanthine) and energy turbulence caused by the intoxication (e.g., increased succinate). To our knowledge, this is the first case of 3-NPA intoxication reported in Norway and the first published metabolomics study of human 3-NPA intoxication worldwide. The unexpected identification of 3-NPA illustrates the importance for health care providers to consider intake-related intoxications during diagnostic evaluations, treatment and follow-up examinations for neurotoxicity and a wide range of metabolic derangements.

Bridging the Polar and Hydrophobic Metabolome in Single-Run Untargeted Liquid Chromatography-Mass Spectrometry Dried Blood Spot Metabolomics for Clinical Purposes.

Dried blood spot (DBS) metabolite analysis is a central tool for the clinic, e.g., newborn screening. Instead of applying multiple analytical methods, a single liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolites spanning from highly polar glucose to hydrophobic long-chain acylcarnitines. For liquid chromatography, a diphenyl column and a multi-linear solvent gradient operated at elevated flow rates allowed for an even-spread resolution of diverse metabolites. Injecting moderate volumes of DBS organic extracts directly, in contrast to evaporation and reconstitution, provided substantial increases in analyte recovery. Q Exactive MS settings were also tailored for sensitivity increases, and the method allowed for analyte retention time and peak area repeatabilities of 0.1-0.4 and 2-10%, respectively, for a wide polarity range of metabolites (log P -4.4 to 8.8). The method's performance was suited for both untargeted analysis and targeted approaches evaluated in clinically relevant experiments.