Endocannabinoid analysis in GlucoEXACT plasma: Method validation and sample handling recommendations.

Endocannabinoids (ECs), such as anandamide and 2-arachidonyl glycerol (2-AG), contribute to the pathology of inflammatory, malignant, cardiovascular, metabolic and mental diseases. The reliability of quantitative analyses in biological fluids of ECs and endocannabinoid-like (EC-like) substances depends on pre-analytical conditions such as temperature and "time-to-centrifugation". Standardization of these parameters is critical for valid quantification and implementation in clinical research. In this study, we compared concentrations obtained with GlucoEXACT blood collection tubes versus K3EDTA tubes and employed the optimized procedure to assess ECs profiles in patients with inflammatory skin disease and healthy controls. A UHPLC-MS/MS method was validated for human plasma from GlucoEXACT blood collection tubes according to EMA and FDA guidelines, and pre-analytical conditions were systematically modified to assess analyte stability and optimize the procedures. The results showed significantly lower concentrations of ECs and EC-like substance concentrations with GlucoEXACT tubes compared with K3EDTA tubes, and GlucoEXACT extended the time window of stable concentrations. The strongest method-disagreement occurred for 1/2-AG suggesting that GlucoEXACT delayed ex vivo isomer rearrangement. Hence, GlucoExact tubes were superior in terms of stability and reliability. However, although absolute concentrations obtained with GlucoExact and K3EDTA differed, linear regression studies showed high agreement (except for 1/2-AG), and both methods showed similar EC profiles and similar disease-dependent pro-inflammatory patterns in dermatology patients. Hence, despite the obstacles in EC analyses, implementation of optimized pre-analytical blood collection and sample processing procedures provide reliable insight into peripheral ECs.

Metabolomic and lipidomic fingerprints in inflammatory skin diseases - Systemic illumination of atopic dermatitis, hidradenitis suppurativa and plaque psoriasis.

Auto-inflammatory skin diseases place considerable symptomatic and emotional burden on the affected and put pressure on healthcare expenditures. Although most apparent symptoms manifest on the skin, the systemic inflammation merits a deeper analysis beyond the surface. We set out to identify systemic commonalities, as well as differences in the metabolome and lipidome when comparing between diseases and healthy controls. Lipidomic and metabolomic LC-MS profiling was applied, using plasma samples collected from patients suffering from atopic dermatitis, plaque-type psoriasis or hidradenitis suppurativa or healthy controls. Plasma profiles revealed a notable shift in the non-enzymatic anti-oxidant defense in all three inflammatory disorders, placing cysteine metabolism at the center of potential dysregulation. Lipid network enrichment additionally indicated the disease-specific provision of lipid mediators associated with key roles in inflammation signaling. These findings will help to disentangle the systemic components of autoimmune dermatological diseases, paving the way to individualized therapy and improved prognosis.

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.

Pre-analytical challenges for the quantification of endocannabinoids in human serum.

Endocannabinoids (ECs) are potent lipid mediators with high physiological relevance. They are involved in a wide variety of diseases like depression or multiple sclerosis and are closely connected to metabolic parameters in humans. Therefore, their suitability as a biomarker in different (patho-)physiological conditions is discussed intensively and predominantly investigated by analyzing systemic concentrations in easily accessible matrices like blood. Carefully designed pre-analytical sample handling is of major importance for high-quality data, but harmonization is not achieved yet. Whole blood is either processed to serum or plasma before the onset of analytical workflows and while knowledge about pre-analytical challenges in plasma handling is thorough they were not systematically investigated for serum. Therefore, the ECs AEA and 2-AG, and closely related EC-like substances 1-AG, DHEA, and PEA were examined by LC-MS/MS in serum samples of nine healthy volunteers employing different pre-analytical sample handling protocols, including prolonged coagulation, and storage after centrifugation at room temperature (RT) or on ice. Furthermore, all analytes were also assessed in plasma samples obtained from the same individuals at the same time points to investigate the comparability between those two blood-based matrices regarding obtained concentrations and their 2-AG/1-AG ratio. This study shows that ECs and EC-like substances in serum samples were significantly higher than in plasma and are especially prone to ex vivo changes during initial and prolonged storage for coagulation at RT. Storage on ice after centrifugation is less critical. However, storage at RT further increases 1-AG and 2-AG concentrations, while also lowering the already reduced 2-AG/1-AG ratio due to isomerization. Thus, avoidance of prolonged processing at RT can increase data quality if serum as the matrix of choice is unavoidable. However, serum preparation in itself is expected to initiate changes of physiological concentrations as standard precautionary measures like fast and cooled processing can only be utilized by using plasma, which should be the preferred matrix for analyses of ECs and EC-like substances.

Determination of endocannabinoids and endocannabinoid-like substances in human K3EDTA plasma - LC-MS/MS method validation and pre-analytical characteristics.

The determination of endocannabinoids and endocannabinoid-like substances in biological human samples is a vibrant field of research with great significance due to postulated relevance of these substances in diseases such as Alzheimer's disease, multiple sclerosis, cancer and cardiovascular diseases. For a possible use as biomarker in early prediction or diagnosis of a disease as well as examination of a successful treatment, the valid determination of the analytes in common accessible human samples, such as plasma or serum, is of great importance. A method for the determination of arachidonoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, 1-arachidonoyl glycerol and 2-arachidonoyl glycerol in human K3EDTA plasma using liquid-liquid-extraction in combination with liquid chromatography-tandem-mass spectrometry has been developed and validated for the quantification of the aforementioned analytes. Particular emphasis was placed on the chromatographic separation of the isomers 1-arachidonoyl glycerol and 2-arachidonoyl glycerol, arachidonoyl ethanolamide and O-arachidonoyl ethanolamine (virodhamine) as well as oleoyl ethanolamide and vaccenic acid ethanolamide. During the validation process, increasing concentrations of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol while storing plasma samples were observed. In-depth investigation of pre-analytical sample handling revealed rising concentrations for both analytes in plasma and for arachidonoyl ethanolamide, oleoyl ethanolamide and palmitoyl ethanolamide in whole blood, dependent on the period and temperature of storage. Prevention of the increase in concentration was not possible, raising the question whether human K3EDTA plasma is suitable for the determination of endocannabinoids and endocannabinoid-like substances. Especially the common practice to calculate the concentration of 2-arachidonoyl glycerol as sum of 1-arachidonoyl glycerol and 2-arachidonoyl glycerol is highly questionable because the concentrations of both analytes increase unequally while storing the plasma samples in the fridge.

Brain cellular localization of endothelin receptors A and B in a rodent model of diffuse traumatic brain injury.

Endothelin-1 exerts potent vasoconstrictor and vasodilatory effects through its actions on its receptors A (ETrA) and B (ETrB), respectively. While ETrA and B have classically been thought to be expressed on vascular cell types, more recent evidence suggests that, particularly following brain injury, their expression may be seen in other, non-vascular cell types. To date no studies have comprehensively studied the cellular location of endothelin receptors following traumatic brain injury (TBI). Therefore, this study investigates the cellular localization of ETrA and B in normal and traumatized brains using an impact acceleration device. Adult male Sprague-Dawley rats were subjected to TBI by weight drop (450 g) from either 1.5, a distance known to elicit mild TBI in the absence of changed in cerebral blood flow (CBF) or 2 m, a distance shown to cause a significant reduction in CBF. One set of impacted brains were processed for Western determination of ETrA and B expression. Another set were processed for immunofluorescence (IF). For IF, ETrA and ETrB antibodies were combined with cell markers for neurons, astrocytes, microglia, oligodendrocytes, smooth muscle cells and endothelial cells of blood vessels. While ETrA and B was upregulated after more moderate to severe injury (2 m) overall receptor expression was unchanged in response to mild trauma (1.5 m). Double labeling IF confirmed prominent ETrA and ETrB labeling in NeuN labeled pyramidal neurons and interneurons in sensorymotor cortex (smCx) and hippocampus (hipp) post TBI. ETrA rather than ETrB was preferentially co-localized in vascular smooth muscle cells. After injury, a subpopulation of astrocytes in white matter co-localized ETrA but not ETrB. Localization of either receptor in endothelial cells was sparse. No prominent IF was detected in microglia and oligodendrocytes. Taken together with previous findings in other pathological states that show an apparent shift in the localization of ETrA and B, the observed receptor shifts in this work may underlie the ET-1-mediated pathotrajectory of TBI including hypoperfusion.

First isolation of Borrelia lusitaniae from a human patient.

The first human isolate of Borrelia lusitaniae recovered from a Portuguese patient with suspected Lyme borreliosis is described. This isolate, from a chronic skin lesion, is also the first human isolate of Borrelia in Portugal. Different phenotypic and molecular methods are used to characterize it.

Association of Borrelia afzelii with rodents in Europe.

Borrelia burgdorferi sensu lato (s.l.) is maintained in nature by complex zoonotic transmission cycles, involving a large variety of vertebrates as hosts and hard ticks of the genus Ixodes as vectors. Recent studies suggest that the genospecies of B. burgdorferi s.l. and sometimes their subtypes are propagated by different spectra of hosts, mainly birds and rodents. In order to test the concept of host-association, we analysed the relationships between Borrelia genospecies, rodent hosts and I. ricinus ticks in an endemic focus of Lyme borreliosis in western Slovakia. Rodents and questing ticks were collected at a forested low land locality near Bratislava. Tick infestation levels on rodents were determined, and spirochaete infections in ticks and in ear punch biopsies were analysed by PCR followed by genotyping. Mice were more heavily infested with ticks than bank voles, and a higher proportion of mice was infected with spirochactes than voles. However, the infectivity of soles was much higher than that of mice. The vast majority of infections detected in the skin and in ticks feeding on the rodents represented B. afzelii. In contrast, more than half of all infections in questing ticks collected in the same region of Slovakia were identified as B. valaisiana and B. garinii. In conclusion, whilst the study reveals that mice and voles play different quantitative roles in the ecology of Lyme borreliosis, it demonstrates that B. afzelii is specifically maintained by European rodents, validating the concept of host-association of B. burgdorferi s.l.

Distinct combinations of Borrelia burgdorferi sensu lato genospecies found in individual questing ticks from Europe.

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.