A comprehensive UHPLC-MS/MS method for metabolomics profiling of signaling lipids: Markers of oxidative stress, immunity and inflammation.

Signaling lipids (SLs) play a crucial role in various signaling pathways, featuring diverse lipid backbone structures. Emerging evidence showing the biological significance and biomedical values of SLs has strongly spurred the advancement of analytical approaches aimed at profiling SLs. Nevertheless, the dramatic differences in endogenous abundances across lipid classes as well as multiple isomers within the same lipid class makes the development of a generic analytical method challenging. A better analytical method that combines comprehensive coverage and high sensitivity is needed to enable us to gain a deeper understanding of the biochemistry of these molecules in health and disease. In this study, we developed a fast and comprehensive targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for profiling SLs. The platform enables analyses of 260 metabolites covering oxylipins (isoprostanes, prostaglandins and other oxidized lipids), free fatty acids, lysophospholipids, sphingoid bases (C16, C18), platelet activating factors (C16, C18), endocannabinoids and bile acids. Various validation parameters including linearity, limit of detection, limit of quantification, extraction recovery, matrix effect, intra-day and inter-day precision were used to characterize this method. Metabolite quantitation was successfully achieved in both NIST Standard Reference Material for human plasma (NIST SRM 1950) and pooled human plasma, with 109 and 144 metabolites quantitated. The quantitation results in NIST SRM 1950 plasma demonstrated good correlations with certified or previously reported values in published literature. This study introduced quantitative data for 37 SLs for the first time. Metabolite concentrations measured in NIST SRM 1950 will serve as essential reference data for facilitating interlaboratory comparisons. The methodology established here will be the cornerstone for in-depth profiling of signaling lipids across diverse biological samples and contexts.

Altered methionine-sulfone levels are associated with impaired growth in HIV-exposed-uninfected children.

OBJECTIVE: To determine immune-metabolic dysregulation in children born to women living with HIV. METHODS: Longitudinal immune-metabolomic analyses of plasma of 32 pregnant women with HIV (WHIV) and 12 uninfected women and their children up to 1.5 years of age were performed. RESULTS: Using liquid chromatography-mass spectrometry and a multiplex bead assay, 280 metabolites (57 amino acids, 116 positive lipids, 107 signalling lipids) and 24 immune mediators (e.g. cytokines) were quantified. combinational antiretroviral therapy (cART) exposure was categorized as cART initiation preconception (long), cART initiation postconception up to 4 weeks before birth (medium) and cART initiation within 3 weeks of birth (short). Plasma metabolite profiles differed between HIV-exposed-uninfected (HEU)-children with long cART exposure compared to HIV-unexposed-children (HUU). Specifically, higher levels of methionine-sulfone, which is associated with oxidative stress, were detected in HEU-children with long cART exposure compared to HUU-children. High infant methionine-sulfone levels were reflected by high prenatal plasma levels in the mother. Increased methionine-sulfone levels in the children were associated with decreased growth, including both weight and length. CONCLUSION: These findings based on longitudinal data demonstrate that dysregulation of metabolite networks associated with oxidative stress in children born to WHIV is associated with restricted infant growth.

Metabolic response of blood vessels to TNFα.

TNFα signaling in the vascular endothelium elicits multiple inflammatory responses that drive vascular destabilization and leakage. Bioactive lipids are main drivers of these processes. In vitro mechanistic studies of bioactive lipids have been largely based on two-dimensional endothelial cell cultures that, due to lack of laminar flow and the growth of the cells on non-compliant stiff substrates, often display a pro-inflammatory phenotype. This complicates the assessment of inflammatory processes. Three-dimensional microvessels-on-a-chip models provide a unique opportunity to generate endothelial microvessels in a more physiological environment. Using an optimized targeted liquid chromatography-tandem mass spectrometry measurements of a panel of pro- and anti-inflammatory bioactive lipids, we measure the profile changes upon administration of TNFα. We demonstrate that bioactive lipid profiles can be readily detected from three-dimensional microvessels-on-a-chip and display a more dynamic, less inflammatory response to TNFα, that resembles more the human situation, compared to classical two-dimensional endothelial cell cultures.

In a range of conditions called autoimmune diseases, the immune system attacks the body rather than foreign elements. This can cause inflammation that is harmful for many organs. In particular, immune cells can produce excessive amounts of a chemical messenger called tumor necrosis factor alpha (TNFα for short), which can lead to the release of fatty molecules that damage blood vessels. This process is normally studied in blood vessels cells that are grown on a dish, without any blood movement. However, in this rigid 2D environment, the cells become 'stressed' and show higher levels of inflammation than in the body. This makes it difficult to assess the exact role that TNFα plays in disease. A new technology is addressing this issue by enabling scientist to culture blood vessels cells in dishes coated with gelatin. This allows the cells to organize themselves in 3D, creating tiny blood vessels in which fluids can flow. However, it was unclear whether these 'microvessels-on-a-chip' were better models to study the role of TNFα compared to cells grown on a plate. Here, Junaid et al. compared the levels of inflammation in blood vessels cells grown in the two environments, showing that cells are less inflamed when they are cultured in 3D. In addition, when the artificial 3D-blood vessels were exposed to TNFα, they responded more like real blood vessels than the 2D models. Finally, experiments showed that it was possible to monitor the release of fatty molecules in this environment. Together, this work suggests that microvessels-on-a-chip are better models to study how TNFα harms blood vessels. Next, systems and protocols could be develop to allow automated mass drug testing in microvessels-on-a-chip. This would help scientists to quickly screen thousands of drugs and find candidates that can protect blood vessels from TNFα.

Persistent metabolic changes in HIV-infected patients during the first year of combination antiretroviral therapy.

The HIV-human metabolic relationship is a complex interaction convoluted even more by antiretroviral therapy (cART) and comorbidities. The ability of cART to undo the HIV induced metabolic dysregulation is unclear and under-investigated. Using targeted metabolomics and multiplex immune biomarker analysis, we characterized plasma samples obtained from 18 untreated HIV-1-infected adult patients and compared these to a non-HIV infected (n = 23) control population. The biogenic amine perturbations during an untreated HIV infection implicated altered tryptophan- nitrogen- and muscle metabolism. Furthermore, the lipid profiles of untreated patients were also significantly altered compared to controls. In untreated HIV infection, the sphingomyelins and phospholipids correlated negatively to markers of infection IP-10 and sIL-2R whereas a strong association was found between triglycerides and MCP-1. In a second cohort, we characterized plasma samples obtained from 28 HIV-1-infected adult patients before and 12 months after the start of cART, to investigate the immune-metabolic changes associated with cART. The identified altered immune-metabolic pathways of an untreated HIV infection showed minimal change after 12 months of cART. In conclusion, 12 months of cART impacts only mildly on the metabolic dysregulation underlying an untreated HIV infection and provide insights into the comorbidities present in virally suppressed HIV patients.

Development and application of a UHPLC-MS/MS metabolomics based comprehensive systemic and tissue-specific screening method for inflammatory, oxidative and nitrosative stress.

Oxidative stress and inflammation are underlying pathogenic mechanisms associated with the progression of several pathological conditions and immunological responses. Elucidating the role of signalling lipid classes, which include, among others, the isoprostanes, nitro fatty acids, prostanoids, sphingoid bases and lysophosphatidic acids, will create a snapshot of the cause and effect of inflammation and oxidative stress at the metabolic level. Here we describe a fast, sensitive, and targeted ultra-high-performance liquid chromatography-tandem mass spectrometry metabolomics method that allows the quantitative measurement and biological elucidation of 17 isoprostanes as well as their respective isomeric prostanoid mediators, three nitro fatty acids, four sphingoid mediators, and 24 lysophosphatidic acid species from serum as well as organ tissues, including liver, lung, heart, spleen, kidney and brain. Application of this method to paired mouse serum and tissue samples revealed tissue- and serum-specific stress and inflammatory readouts. Little correlation was found between localized (tissue) metabolite levels compared with the systemic (serum) circulation in a homeostatic model. The application of this method in future studies will enable us to explore the role of signalling lipids in the metabolic pathogenicity of stress and inflammation during health and disease.

Fetal Metabolic Stress Disrupts Immune Homeostasis and Induces Proinflammatory Responses in Human Immunodeficiency Virus Type 1- and Combination Antiretroviral Therapy-Exposed Infants.

Increased morbidity and fetal growth restriction are reported in uninfected children born to human immunodeficiency virus type 1 (HIV-1)-infected women treated with antiretroviral (ARV) therapy. Viruses and/or pharmacological interventions such as ARVs can induce metabolic stress, skewing the cell's immune response and restricting (cell) growth. Novel metabolomic techniques provided the opportunity to investigate the impact of fetal HIV-1 and combination ARV therapy (cART) exposure on the infants' immune metabolome. Peroxidized lipids, generated by reactive oxygen species, were increased in cART/HIV-1-exposed infants, indicating altered mitochondrial functioning. The lipid metabolism was further dysregulated with increased triglyceride species and a subsequent decrease in phospholipids in cART/HIV-1-exposed infants compared to control infants. Proinflammatory immune mediators, lysophospholipids as well as cytokines such as CXCL10 and CCL3, were increased whereas anti-inflammatory metabolites from the cytochrome P450 pathway were reduced in cART/HIV-1-exposed infants. Taken together, these data demonstrate that the fetal metabolism is impacted by maternal factors (cART and HIV-1) and skews physiological immune responses toward inflammation in the newborn infant.

Ultra-weak photon emission as a dynamic tool for monitoring oxidative stress metabolism.

In recent years, excessive oxidative metabolism has been reported as a critical determinant of pathogenicity in many diseases. The advent of a simple tool that can provide a physiological readout of oxidative stress would be a major step towards monitoring this dynamic process in biological systems, while also improving our understanding of this process. Ultra-weak photon emission (UPE) has been proposed as a potential tool for measuring oxidative processes due to the association between UPE and reactive oxygen species. Here, we used HL-60 cells as an in vitro model to test the potential of using UPE as readout for dynamically monitoring oxidative stress after inducing respiratory burst. In addition, to probe for possible changes in oxidative metabolism, we performed targeted metabolomics on cell extracts and culture medium. Lastly, we tested the effects of treating cells with the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Our results show that UPE can be used as readout for measuring oxidative stress metabolism and related processes.

Metabolomics profiling of the free and total oxidised lipids in urine by LC-MS/MS: application in patients with rheumatoid arthritis.

Oxidised lipids, covering enzymatic and auto-oxidation-synthesised mediators, are important signalling metabolites in inflammation while also providing a readout for oxidative stress, both of which are prominent physiological processes in a plethora of diseases. Excretion of these metabolites via urine is enhanced through the phase-II conjugation with glucuronic acid, resulting in increased hydrophilicity of these lipid mediators. Here, we developed a bovine liver-ß-glucuronidase hydrolysing sample preparation method, using liquid chromatography coupled to tandem mass spectrometry to analyse the total urinary oxidised lipid profile including the prostaglandins, isoprostanes, dihydroxy-fatty acids, hydroxy-fatty acids and the nitro-fatty acids. Our method detected more than 70 oxidised lipids biosynthesised from two non-enzymatic and three enzymatic pathways in urine samples. The total oxidised lipid profiling method was developed and validated for human urine and was demonstrated for urine samples from patients with rheumatoid arthritis. Pro-inflammatory mediators PGF2α and PGF3α and oxidative stress markers iPF2α- IV, 11-HETE and 14-HDoHE were positively associated with improvement of disease activity score. Furthermore, the anti-inflammatory nitro-fatty acids were negatively associated with baseline disease activity. In conclusion, the developed methodology expands the current metabolic profiling of oxidised lipids in urine, and its application will enhance our understanding of the role these bioactive metabolites play in health and disease.

Metabolic characterization of the natural progression of chronic hepatitis B.

BACKGROUND: Worldwide, over 350 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing progressive liver diseases. The confinement of HBV replication to the liver, which also acts as the central hub for metabolic and nutritional regulation, emphasizes the interlinked nature of host metabolism and the disease. Still, the metabolic processes operational during the distinct clinical phases of a chronic HBV infection-immune tolerant, immune active, inactive carrier, and HBeAg-negative hepatitis phases-remains unexplored. METHODS: To investigate this, we conducted a targeted metabolomics approach on serum to determine the metabolic progression over the clinical phases of chronic HBV infection, using patient samples grouped based on their HBV DNA, alanine aminotransferase, and HBeAg serum levels. RESULTS: Our data illustrate the strength of metabolomics to provide insight into the metabolic dysregulation experienced during chronic HBV. The immune tolerant phase is characterized by the speculated viral hijacking of the glycerol-3-phosphate-NADH shuttle, explaining the reduced glycerophospholipid and increased plasmalogen species, indicating a strong link to HBV replication. The persisting impairment of the choline glycerophospholipids, even during the inactive carrier phase with minimal HBV activity, alludes to possible metabolic imprinting effects. The progression of chronic HBV is associated with increased concentrations of very long chain triglycerides together with citrulline and ornithine, reflective of a dysregulated urea cycle peaking in the HBV envelope antigen-negative phase. CONCLUSIONS: The work presented here will aid in future studies to (i) validate and understand the implication of these metabolic changes using a thorough systems biology approach, (ii) monitor and predict disease severity, as well as (iii) determine the therapeutic value of the glycerol-3-phosphate-NADH shuttle.

Collagen Induced Arthritis in DBA/1J Mice Associates with Oxylipin Changes in Plasma.

Oxylipins play important roles in various biological processes and are considered as mediators of inflammation for a wide range of diseases such as rheumatoid arthritis (RA). The purpose of this research was to study differences in oxylipin levels between a widely used collagen induced arthritis (CIA) mice model and healthy control (Ctrl) mice. DBA/1J male mice (age: 6-7 weeks) were selected and randomly divided into two groups, namely, a CIA and a Ctrl group. The CIA mice were injected intraperitoneally (i.p.) with the joint cartilage component collagen type II (CII) and an adjuvant injection of lipopolysaccharide (LPS). Oxylipin metabolites were extracted from plasma for each individual sample using solid phase extraction (SPE) and were detected with high performance liquid chromatography/tandem mass spectrometry (HPLC-ESI-MS/MS), using dynamic multiple reaction monitoring (dMRM). Both univariate and multivariate statistical analyses were applied. The results in univariate Student's t-test revealed 10 significantly up- or downregulated oxylipins in CIA mice, which were supplemented by another 6 additional oxylipins, contributing to group clustering upon multivariate analysis. The dysregulation of these oxylipins revealed the presence of ROS-generated oxylipins and an increase of inflammation in CIA mice. The results also suggested that the collagen induced arthritis might associate with dysregulation of apoptosis, possibly inhibited by activated NF-κB because of insufficient PPAR-γ ligands.

Fatal disseminated toxoplasmosis in an immunocompetent cat.

A 10-year-old domestic short hair cat was referred for investigation of anorexia and polydipsia of 3 days' duration. Clinically the cat was obese, pyrexic (39.8 °C), had acute abdominal pain and severe bilirubinuria. Haematology and serum biochemistry revealed severe panleukopenia, thrombocytopenia, markedly elevated alanine aminotransferase (ALT) and five-fold increased pre-prandial bile acids. Ultrasonographic evaluation of the abdomen did not identify any abnormalities. Serum tests for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) were negative. Broad-spectrum antibiotic treatment for infectious hepatitis was to no avail; the cat deteriorated and died 72 h after admission. Necropsy revealed mild icterus and anaemia, severe multifocal hepatic necrosis, serofibrinous hydrothorax, pulmonary oedema and interstitial pneumonia. Histopathology confirmed the macroscopic findings and revealed multifocal microgranulomata in the brain and myocardium, as well as areas of necrosis in lymph nodes and multifocally in splenic red pulp. Long bone shaft marrow was hyperplastic with a predominance of leukocyte precursors and megakaryocytes and splenic red pulp showed mild extramedullary haemopoiesis. Immunohistochemical staining for Toxoplasma gondii was strongly positive, with scattered cysts and tachyzoites in the liver, lymph nodes, spleen, lungs, brain, salivary glands and intracellularly in round cells in occasional blood vessels. Immunohistochemical staining for corona virus on the same tissues was negative, ruling out feline infectious peritonitis (FIP). Polymerase chain reaction (PCR) on formalin-fixed paraffin-wax embedded tissues was positive for Toxoplasma sp., but attempts at sequencing were unsuccessful. This was the first case report of fulminant disseminated toxoplasmosis in South Africa, in which detailed histopathology in an apparently immunocompetent cat was described.

A comparison of four sputum pre-extraction preparation methods for identifying and characterising Mycobacterium tuberculosis using GCxGC-TOFMS metabolomics.

In many pulmonary diseases, sputum is a valuable sample material for use in disease characterisation and diagnostics. However, due to its high viscosity and uneven consistency (lumpiness), it is difficult to obtain reproducible/repeatable results during compound extraction and analysis. We subsequently investigated and compared four sputum pre-extraction preparation methods using: 1) Sputolysin; 2) a combination of N-acetyl-l-cysteine and sodium hydroxide (NALC-NaOH); 3) NaOH alone, and 4) a simple ethanol homogenisation method, prior to sputum extraction and metabolomics analyses. The simple ethanol homogenisation approach proved to be the comparatively superior sputum pre-extraction preparation method, considering its repeatability, the number of characteristic compounds extracted, its ability to extract those compounds best differentiating the sample groups (Mycobacterium tuberculosis-spiked and clinically confirmed TB-positive patient samples from each of the controls respectively), and its detection limit. This developed methodology subsequently allows for accurate GC based analyses of sputum, and hence, could contribute significantly to the better characterisation or diagnostics of not only tuberculosis, but also potentially other pulmonary diseases, including, interstitial lung disease, cystic fibrosis, lung cancer, pneumonia and any other bacterial induced pulmonary diseases producing sputum.

Capillary and venous Babesia canis rossi parasitaemias and their association with outcome of infection and circulatory compromise.

This observational study of 100 dogs naturally infected with Babesia canis rossi determined whether severity of parasitaemia was associated with outcome of infection and documented the relative distribution of parasitised red blood cells (pRBC) in capillary and venous circulation. The association between increased parasitaemias and outcome with a clinically compromised circulation was also investigated. Outcome was defined as either hospitalisation with death, or hospitalisation with eventual recovery or treatment as an outpatient. Dogs were enrolled if large babesias were found on stained thin capillary blood smears made from an ear prick. Thin venous smears were prepared from jugular or cephalic blood. Parasitaemias were manually counted and expressed as the percent pRBC. Ten dogs died, 50 recovered after hospitalisation and 40 were treated as outpatients. Venous sampling site did not affect venous parasitaemia (P=0.6). Both capillary and venous parasitaemias of dogs that died were significantly higher than those of dogs that recovered after hospitalisation (P=0.002) and dogs that were treated as outpatients (P<0.0001). When assessing the whole group, capillary parasitaemia (median 0.61%, range <0.05-71.6%, interquartile range (IQR) 0.22-3.75%) was significantly higher than venous parasitaemia (median 0.14%, range 0-30.6%, IQR 0.046-0.52%) with P<0.0001. The 21 dogs with a clinically compromised circulation were more likely to die (P<0.0001) and had significantly higher capillary (median 5.98%, range 0.09-71.6%, IQR 2.44-19.41%) and venous (median 2.81%, range <0.05-30.6%, IQR 0.17-9.03%) parasitaemias than the 79 dogs with a clinically normal circulation (capillary median parasitaemia 0.38%, range <0.05-12.87%, IQR 0.16-1.42%; venous median parasitaemia 0.096%, range 0-6.13%, IQR <0.05-0.33%; P<0.0001). This study shows that high parasitaemia is significantly associated with death in B c rossi infected dogs. The previous clinical suspicion that capillary parasitaemias are usually higher than venous parasitaemias is confirmed. Thus capillary samples are the most appropriate diagnostic samples. Prior observations that a clinically compromised circulation is associated with death are confirmed. Despite the highly significant association between compromised circulation and higher parasitaemia, it is thought unlikely that parasite burden is the sole trigger for circulatory collapse.