Discovery and Verification of Sjögren's Syndrome Protein Biomarkers in Tears by Targeted LC-MRM.

Sjögren's syndrome (SS) is an autoimmune rheumatic disorder characterized by exocrine gland dysfunction, mainly from the lacrimal and salivary glands. The disease causes severe aqueous dry eye syndrome (DED) and is associated with high rates of complications, including corneal ulceration, scaring, and perforation. Systemic complications may occur as well as a higher risk of developing lymphoma. Diagnosis of SS-DED is often delayed and difficult to establish. With the aim of discovering biomarkers to help discriminate SS-DED patients, a combination of untargeted and targeted LC-MS/MS analyses were performed on tear samples collected on Schirmer strips and subjected to tryptic digestion. Following the analysis of three cohorts and the development of two targeted LC-sMRM methods for the verification of putative biomarkers found in the first cohort of samples, 64 proteins could be linked to Sjögren's syndrome, in the hopes of helping to confirm diagnoses as well as potentially stratifying the severity of disease in these patients. Proteins that were increased in SS-DED showed activation of the immune system and alterations in homeostasis. Several proteases and protease inhibitors were found to be significantly changing in SS-DED, as well as a consistent decrease in specific proteins known to be secreted by the lacrimal gland.

Multiple ammonia-induced episodes of hepatic encephalopathy provoke neuronal cell loss in bile-duct ligated rats.

Background & Aims: Hepatic encephalopathy (HE) is defined as a reversible syndrome and therefore should resolve following liver transplantation (LT). However, neurological complications have been reported in up to 47% of LT recipients, which have been documented to be associated with a history of overt HE pre-LT. We hypothesise that multiple episodes of HE lead to permanent cell injury and exacerbate neurological dysfunction. Our goal was to evaluate the impact of cumulative HE episodes on neurological status and brain integrity in rats with chronic liver disease. Methods: Episodes of overt HE (loss of righting reflex) were induced following injection of ammonium acetate in bile duct ligation (BDL) rats (BDL-Ammonia) every 4 days starting at week 3 post-BDL. Neurobehaviour was evaluated after the last episode. Upon sacrifice, plasma ammonia, systemic oxidative stress, and inflammation markers were assessed. Neuronal markers including neuron-specific nuclear antigen and SMI311 (anti-neurofilament marker) and apoptotic markers (cleaved caspase-3, Bax, and Bcl2) were measured. Total antioxidant capacity, oxidative stress marker (4-hydroxynonenal), and proinflammatory cytokines (tumour necrosis factor-alpha and interleukin-1ß) were measured in brain (hippocampus, frontal cortex, and cerebellum). Proteomic analysis was conducted in the hippocampus. Results: In hippocampus of BDL-Ammonia rats, cleaved caspase-3 and Bax/Bcl2 ratio were significantly increased, whereas NeuN and SMI311 were significantly decreased compared with BDL-Vehicle rats. Higher levels of oxidative stress-induced post-translational modified proteins were found in hippocampus of BDL-Ammonia group which were associated with a lower total antioxidant capacity. Conclusions: Ammonia-induced episodes of overt HE caused neuronal cell injury/death in BDL rats. These results suggest that multiple bouts of HE can be detrimental on the integrity of the brain, translating to irreversibility and hence neurological complications post-LT. Impact and implications: Hepatic encephalopathy (HE) is defined as a reversible neuropsychiatric syndrome resolving following liver transplantation (LT); however, ∼47% of patients demonstrate neurological impairments after LT, which are associated with a previous history of overt HE pre-LT. Our study indicates that multiple episodes of overt HE can cause permanent neuronal damage which may lead to neurological complications after LT. Nevertheless, preventing the occurrence of overt HE episodes is critical for reducing the risk of irreversible neuronal injury in patients with cirrhosis.

Targeted Workflow Investigating Variations in the Tear Proteome by Liquid Chromatography Tandem Mass Spectrometry.

Proteins in tears have an important role in eye health and have been shown as a promising source of disease biomarkers. The goal of this study was to develop a robust, sensitive, and targeted method for profiling tear proteins to examine the variability within a group of healthy volunteers over three days. Inter-individual and inter-day variabilities were examined to contribute to understanding the normal variations in the tear proteome, as well as to establish which proteins may be better candidates as eventual biomarkers of specific diseases. Tear samples collected on Schirmer strips were subjected to bottom-up proteomics, and resulting peptides were analyzed using an optimized targeted method measuring 226 proteins by liquid chromatography-scheduled multiple reaction monitoring. This method was developed using an in-house database of identified proteins from tears compiled from high-resolution data-dependent liquid chromatography tandem mass spectrometry data. The measurement of unique peptide signals can help better understand the dynamics of each of these proteins in tears. Some interesting trends were seen in specific pathways or protein classes, including higher variabilities for those involved in glycolysis, glutathione metabolism, and cytoskeleton proteins and lower variation for those involving the degradation of the extracellular matrix. The overall aim of this study was to contribute to the field of tear proteomics with the development of a novel and targeted method that is highly amenable to the clinical laboratory using high flow LC and commonly used triple quadrupole mass spectrometry while ensuring that protein quantitation was reported based on unique peptides for each protein and robust peak areas with data normalization. These results report on variabilities on over 200 proteins that are robustly detected in tear samples from healthy volunteers with a simple sample preparation procedure.

A validated UPLC-MS/MS method for the determination of aliphatic and aromatic isocyanate exposure in human urine.

4,4'-Methylenediphenyldiisocyanate (MDI), toluenediisocyanate (2,4-TDI and 2,6-TDI), and 1,6'-hexamethylenediisocyanate (HDI) are all commonly used in the production of polyurethane-containing materials in different application areas. Workers exposed occupationally to these compounds may develop sensitization with the potential to lead to asthma. Isocyanates are metabolized in vivo by conjugation to macromolecules and/or by acetylation prior to being eliminated in urine. The hydrolysis of urine samples releases free amine compounds from these metabolites as biomarkers of exposure, specific to each parent isocyanate: 4,4'-methylenedianiline (MDA), toluenediamine (2,4-TDA and 2,6-TDA), and hexamethylenediamine (HDA). To address the need for a validated method that could be used for the simultaneous determination of biomarkers of aliphatic and aromatic isocyanates to monitor occupational exposure based on recommended thresholds, we have developed an UPLC-MS/MS method for the quantitation of MDA, TDA isomers, and HDA following acid hydrolysis, solid-phase extraction, and derivatization of urine samples. Free amine compounds were derivatized with acetic anhydride to augment chromatographic retention and signal intensity. The method was developed considering the biological guidance value (BGV) of MDA at 10 µg L-1, and biological exposure indices (BEI) of TDA isomers and HDA at 5 µg g-1 and 15 µg g-1 creatinine, respectively. Limits of detection allowed monitoring down to 6% of BGV/BEI, with precision within 8%. The accuracy and reliability of the method were assessed using inter-laboratory reference samples and deemed acceptable based on three rounds of measurements. This novel method has therefore been proven as useful for occupational safety and health assessments. Graphical Abstract.

A validated liquid chromatography/tandem mass spectrometry method for 4,4'-methylenedianiline quantitation in human urine as a measure of 4,4'-methylene diphenyl diisocyanate exposure.

RATIONALE: 4,4'-Methylene diphenyl diisocyanate (MDI) is a highly reactive isocyanate used in the production of polyurethanes. Workers exposed to these products may develop sensitization to the diisocyanate compounds, leading to occupational asthma. Quantifying MDI levels is necessary to ensure workplace safety. MDI is metabolized by acetylation and/or conjugation to macromolecules for excretion into urine. All metabolites can be chemically hydrolyzed to form the free diamine 4,4'-methylenedianiline (MDA) as a urinary biomarker of MDI exposure. Current methods involve long sample preparation, or have been designed using costly automation. There is therefore a need to develop a new practical method for assessing exposure to MDI. METHODS: Urine samples were acidified and heated to form MDA, followed by neutralization and liquid-liquid extraction. Extracts were separated by reversed-phase chromatography on a HSS T3 column followed by analysis on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. RESULTS: 13 C15 N-MDA was selected as the internal standard (IS) of choice following an investigation of internal standard stability. The hydrolysis efficiency, forming free MDA from conjugated metabolites in vivo, was evaluated using 4,4'-methylenebis(acetanilide) spiked into urine and complete hydrolysis occurred after 1 h. A dynamic range of 5 to 500 nM was achieved, and was useful for monitoring MDI exposure considering the biological guidance value (BGV) of 10 µg/L (~50 nM) proposed by the German Research Foundation (DFG). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.8 and 2.7 nM, respectively. The intra-day and inter-day precisions were 4.33% and 4.27%, respectively. Finally, the method was tested with inter-laboratory samples from the German External Quality Assessment Scheme (G-EQUAS) program and the results submitted were all within the allowable tolerance range. CONCLUSIONS: A practical and validated method for the analysis of small- to medium-sized batches of samples has been developed for the biological monitoring of MDI exposure in human urine.