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.

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics. Targeted analyses of small molecules are the main application of dried blood spot samples and liquid chromatography-mass spectrometry, but emerging applications include untargeted metabolomics and proteomics. Applications are highly varied, including analyses related to newborn screening, diagnostics and monitoring of disease progression and treatment effects of virtually any disease, as well as studies into the physiology and effects of diet, exercise, xenobiotics, and doping. A range of dried blood spot products and methods are available, and applied liquid chromatography-mass spectrometry instrumentation is varied with regard to liquid chromatography column formats and selectivity. In addition, novel approaches such as on-paper sample preparation (e.g., selective trapping of analytes with paper-immobilized antibodies) are described. We focus on research papers published in the last 5 years.

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.

Increased Sphingomyelin and Free Sialic Acid in Cerebrospinal Fluid of Kearns-Sayre Syndrome: New Findings Using Untargeted Metabolomics.

BACKGROUND: Kearns-Sayre syndrome (KSS) is caused by duplications and/or deletions of mitochondrial DNA (mtDNA) and is typically diagnosed based on a classic triad of symptoms with chronic progressive external ophthalmoplegia (CPEO), retinitis pigmentosa, and onset before age 20 years. The present study aimed to diagnose two patients, on suspicion of KSS. METHODS: One of the patients went through a diagnostic odyssey, with normal results from several mtDNA analyses, both in blood and muscle, before the diagnosis was confirmed genetically. RESULTS: Two patients presented increased tau protein and low 5-methyltetrahydrofolate (5-MTHF) levels in the cerebrospinal fluid (CSF). Untargeted metabolomics on CSF samples also showed an increase in the levels of free sialic acid and sphingomyelin C16:0 (d18:1/C16:0), compared with four control groups (patients with mitochondrial disorders, nonmitochondrial disorders, low 5-MTHF, or increased tau proteins). CONCLUSIONS: It is the first time that elevated sphingomyelin C16:0 (d18:1/C16:0) and tau protein in KSS are reported. Using an untargeted metabolomics approach and standard laboratory methods, the study could shed new light on metabolism in KSS to better understand its complexity. In addition, the findings may suggest the combination of elevated free sialic acid, sphingomyelin C16:0 (d18:1/C16:0), and tau protein as well as low 5-MTHF as new biomarkers in the diagnostics of KSS.

A global metabolomics minefield: Confounding effects of preanalytical factors when studying rare disorders.

A common challenge when studying rare diseases or medical conditions is the limited number of patients, usually resulting in long inclusion periods as well as unequal sampling and storage conditions. The main purpose of this study was to demonstrate the challenges when comparing samples subject to different preanalytical conditions. We performed a global (commonly referred to as "untargeted") liquid chromatography-high resolution mass spectrometry metabolomics analysis of blood samples from cases of sudden infant death syndrome and controls stored as dried blood spots on a chemical-free filter card for 15 years at room temperature compared with the same blood samples stored as whole blood at -80°C before preparing new dried blood spots using a chemically treated filter card. Principal component analysis plots distinctly separated the samples based on the type of filter card and storage, but not sudden infant death syndrome versus controls. Note that, 1263 out of 5161 and 642 out of 1587 metabolite features detected in positive and negative ionization mode, respectively, were found to have significant 2-fold changes in amounts corresponding to different preanalytical conditions. The study demonstrates that the dried blood spot metabolome is largely affected by preanalytical factors. This emphasizes the importance of thoroughly addressing preanalytical factors during study design and interpretation, enabling identification of real, biological differences between sample groups whilst preventing other factors or random variation to be falsely interpreted as positive results.

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.

High Plasma Oxalate Levels Early After Kidney Transplantation Are Associated With Impaired Long-Term Outcomes.

Background: Elevated levels of oxalate are common in renal failure patients and non-hyperoxaluria disease, and may cause damage after transplantation. We examined outcomes after 15 years for 167 kidney transplant recipients who had plasma oxalate measured early after transplantation. Analyses included plasma oxalate, recipient age, donor age, live donor, HLA-DR mismatch, mGFR, and smoking. Results: Median age was 52 years (range 18-81), 63% were male and 38% had live donors. Median plasma oxalate concentration 10 weeks after transplantation was 9.0 µmol/L (range 2.7-53.0), one third above the upper reference limit (11.0 µmol/L). Multivariable analysis revealed upper quartile plasma oxalate (>13.0 µmol/L, p = 0.008), recipient age (p < 0.001), deceased donor (p = 0.003), and current smoking (p < 0.001) as significant factors associated with patient survival. Upper quartile plasma oxalate (p = 0.021), recipient age (p = 0.001), deceased donor kidney (p = 0.001), HLA-DR mismatch (p = 0.015), and current smoking (p = 0.014) were also associated with graft loss. Factors associated with death censored graft losses were donor age (p = 0.012), deceased donor (p = 0.032), and HLA-DR mis-matched kidneys (p = 0.005) but plasma oxalate was not (p = 0.188). Conclusions: Plasma oxalate in the upper quartile early after transplantation was significantly associated with impaired long-term patient survival and graft losses, but not when censored for death.

Metabolomics - a new biochemical golden age for personalised medicine. / Metabolomikk ­ ny biokjemisk gullalder for persontilpasset medisin.

Thousands of different molecules can be measured in one drop of blood. Metabolomics can detect 'all' the small molecular substances in any sample material. This is the biochemistry equivalent of whole genome sequencing in genetics. The technology can take personalised medicine to the next level.

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.

Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.

Purine metabolism is essential for all known living creatures, including humans in whom elevated serum concentration of purine break-down product uric acid (UA) is probably an independent risk factor for mortality, type 2 diabetes and cardiovascular events. An automated multiplex assay that measures several purine metabolites could therefore prove useful in many areas of medical, veterinary and biological research. The aim of the present work was to develop a sensitive LC-MS/MS method for simultaneous quantitation of xanthine, hypoxanthine, UA, allantoin, and creatinine in biobanked urine samples. This article describes details and performance of the new method studied in 55 samples of human urine. Archival sample preparation and effect of storage conditions on stability of the analytes are addressed. The intra-day and inter-day coefficients of variation were small for all the analytes, not exceeding 1% and 10%, respectively. Measurements of UA and creatinine in biobanked urine showed good agreement with values obtained using routine enzymatic assays on fresh urine. Spearman's correlation coefficients were 0.869 (p < .001) for creatinine and 0.964 (p < .001) for UA. Conclusion: the newly developed LC-MS/MS method allows reliable quantitative assessment of xanthine, hypoxanthine, allantoin, UA and creatinine. The proposed pre-analytical processing makes the method suitable for both fresh and biobanked urine stored frozen at -80 °C for at least 5.5 years.

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.

Eye Make-up Products and Dry Eye Disease: A Mini Review.

Dry eye disease (DED), a multifactorial condition of the tear film and ocular surface, is one of the leading reasons for patients seeking eye care. Despite the multiple toxic ingredients of eye make-up products and their long-term application close to the ocular surface, few studies have analyzed their role in initiating and worsening DED. Females and the elderly experience the highest prevalence of DED and may be particularly vulnerable to the effects of eye make-up. The multifactorial nature of DED and common mechanisms behind several ocular surface diseases make it difficult to link a particular ingredient-driven mechanism to DED. Therefore, here, we list potential responses to eye cosmetics that may be involved in DED development. The first part of this review introduces the anatomy of the eye and DED, the second section explains the classification of eye cosmetic products, and the final part discusses the undesired effects under physical, pathogenic, and chemical insults.

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.

Is oxidative stress an overlooked player in pyridoxine-dependent epilepsy? A focused review.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive developmental and epileptic encephalopathy that is responsive to pharmacologic doses of vitamin B6. The deficiency of antiquitin, an enzyme involved in the catabolism of lysine, is believed to be its key molecular basis. Research to date has tended to focus on two known catabolic pathways of lysine, namely, saccharopine and pipecolic acid. However, the occurrence of oxidative stress and the presence of its metabolites have been only briefly highlighted in the literature. Owing to the importance of the topic and its potential for future diagnosis, prognosis and therapy, this paper reviews the suggested mechanisms of oxidative stress in antiquitin deficiency along with the proposed reactions and intermediates, and finally, discusses the challenges and opportunities.

Searching for a UV-filter in the eyes of high-flying birds.

The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as "compound X", was fractionated and examined using a variety of spectroscopic techniques including LC-MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective "compound X". It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.

Effects of nutrition therapy on growth, inflammation and metabolism in immature infants: a study protocol of a double-blind randomized controlled trial (ImNuT).

BACKGROUND: Current nutritional management of infants born very preterm results in significant deficiency of the essential fatty acids (FAs) arachidonic acid (ARA) and docosahexaenoic acid (DHA). The impact of this deficit on brain maturation and inflammation mediated neonatal morbidities are unknown. The aim of this study is to determine whether early supply of ARA and DHA improves brain maturation and neonatal outcomes in infants born before 29 weeks of gestation. METHODS: Infants born at Oslo University Hospital are eligible to participate in this double-blind randomized controlled trial. Study participants are randomized to receive an enteral FA supplement of either 0.4 ml/kg MCT-oil™ (medium chain triglycerides) or 0.4 ml/kg Formulaid™ (100 mg/kg of ARA and 50 mg/kg of DHA). The FA supplement is given from the second day of life to 36 weeks' postmenstrual age (PMA). The primary outcome is brain maturation assessed by Magnetic Resonance Imaging (MRI) at term equivalent age. Secondary outcomes include quality of growth, incidence of neonatal morbidities, cardiovascular health and neuro-development. Target sample size is 120 infants (60 per group), this will provide 80% power to detect a 0.04 difference in mean diffusivity (MD, mm2/sec) in major white matter tracts on MRI. DISCUSSION: Supplementation of ARA and DHA has the potential to improve brain maturation and reduce inflammation related diseases. This study is expected to provide valuable information for future nutritional guidelines for preterm infants. TRIAL REGISTRATION: Clinicaltrials.gov ID: NCT03555019 . Registered 4 October 2018- Retrospectively registered.

Tear Metabolomics in Dry Eye Disease: A Review.

Dry eye disease (DED) is a multifactorial syndrome that can be caused by alteration in the quality or quantity of the precorneal tear film. It is considered one of the most common ocular conditions leading patients to seek eye care. The current method for diagnostic evaluations and follow-up examinations of DED is a combination of clinical signs and symptoms determined by clinical tests and questionnaires, respectively. The application of powerful omics technologies has opened new avenues toward analysis of subjects in health and disease. Metabolomics is a new emerging and complementary research discipline to all modern omics in the comprehensive analysis of biological systems. The identification of distinct metabolites and integrated metabolic profiles in patients can potentially inform clinicians at an early stage or during monitoring of disease progression, enhancing diagnosis, prognosis, and the choice of therapy. In ophthalmology, metabolomics has gained considerable attention over the past decade but very limited such studies have been reported on DED. This paper aims to review the application of tear metabolomics in DED.

Liquid chromatography-tandem mass spectrometry method for routine measurement of oxalic acid in human plasma.

A solid phase extraction (SPE)-LC-MSMS method for the routine determination of oxalic acid (OX) in plasma, a diagnostic marker of primary hyperoxaluria (PH), was developed and validated. The normal range of OX was found to be 3-11 micromol/L (n=67), with no differences attributable to gender or age. The effect of pre-analytical factors on the in vitro production of OX was investigated, and plasma was found to be stable for 1-2 h at room temperature, less after ingestion of vitamin C; the process was not completely stopped by preservation at either -20 or -70 degrees C.