Cherry-red plasma: Beyond the assumption of hemolysis.

Hemolysis is the most prevalent pre-analytical interfering factor and a major source of error in laboratory analysis. The examination of samples post-centrifugation can provide valuable information regarding pre-analytical interferences. In this unusual case, a patient's plasma specimen was cherry-red after centrifugation, which is most usually indicative of hemolysis. However, subsequent investigations ruled out common hemolysis causes. We eventually determined that the patient's cherry-red plasma was most likely caused by other factors in the patient's medical history, including cancer treatment with PV-10 (rose bengal disodium 10%). We then conducted an interference study to comprehensively assess the effects of PV-10 on various biochemical tests, especially liver function tests and bilirubin levels. The findings indicate that PV-10 has varying effects on different biochemical assays and test results should be examined individually. This report underlines the need for awareness of potential drug interference on laboratory tests for better result interpretation and making clinical decisions.

Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis

ABSTRACT Purpose: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. Methods: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. Results: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. Conclusion: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

Impact of blood volume, air exposure duration, transport duration, and testing delay on plasma total carbon dioxide in simulated open collections using microtainers.

BACKGROUND: Exposing blood specimens to air reduces plasma total carbon dioxide (TCO2). We evaluated the degree of TCO2 reduction attributed to open collection of neonatal blood in BD microtainers® (microtainers), microtainer transport duration and delayed testing of open plasma aliquots. METHODS: Venous blood was aliquoted into open microtainers in a 3x4 factorial design to simulate combined effects of blood volume (0.2-0.6 mL) and air exposure duration (0-5 min), with blood drawn in vacutainers as a control. Separate effects of in-hospital transport duration (0-120 min; whole blood), off-site transport duration (0-24 h; centrifuged whole blood), and the duration plasma aliquots remained open (0-120 min) were evaluated by repeated testing. Findings were analyzed using repeated-measures ANOVA and Student's T-tests. RESULTS: In the factorial experiment, mean plasma TCO2 in microtainers was on average 3.5 mmol/L lower than in vacutainers. Smaller blood volume but not greater air exposure duration significantly (p < 0.05) reduced TCO2. Mean TCO2 in filled (0.6 mL; 1-5 min air exposure) microtainers was on average 2.9 mmol/L lower than in vacutainers. Simulated off-site transport of microtainers containing centrifuged whole blood significantly reduced TCO2 (4 h; mean change = -1.5 mmol/L), as did delayed testing of aliquoted plasma (15 min; mean change = -1.3 mmol/L). CONCLUSIONS: Plasma TCO2 decreased with reduced microtainer blood volume, extended off-site transport duration of centrifuged whole blood and testing delay of aliquoted plasma. To minimize TCO2 reduction, microtainers should be fully filled and tested rapidly. Laboratories should also consider whether an interpretive comment, correction factor or separate reference intervals are appropriate for these tests.

Microscopic mechanistic study of the penetration distributions for plasma reactive oxygen and nitrogen species based on sialic acid targeting on the cell membrane surface.

The ability of cold atmospheric plasmas (CAPs) to produce a wide range of active constituents while maintaining a low or even room temperature of the gas has made it a novel research area of great interest. During plasma action, cancer cell membrane surface components are susceptible to oxidative modification by reactive oxygen and nitrogen species (RONS). In this study, the process of oxidative modification of membrane surface components sialic acid by RONS was investigated based on molecular dynamics simulations, and the penetration mechanism of long-lived particles ONOOH and its homolytic products at the membrane-water interface and the effect of appropriate electric field action were studied. The results showed that cancer cells with high sialic acid expression were less stable than healthy cells. Plasma treatment may promote the ONOOH homolysis process, and its homolysis product OH free radical is more likely to adsorb near sialic acid molecules by hydrogen bonding, resulting in oxidative modification. The interaction force between OH free radical and sialic acid molecules is stronger than ONOOH, which helps to further understand the oxidative modification reaction in membrane environment. At the same time, appropriate electric field stimulation can enhance the depth of penetration of RONS to more effectively treat the pathological state of biological tissues. The study proposes the use of membrane surface sialic acid as a cancer therapeutic target and provides guidance for improving the depth of RONS penetration and maximizing the survival of healthy cells, which contributes to the further clinical translation of plasma biomedicine.

Cold Exposure Alleviates T2DM Through Plasma-Derived Extracellular Vesicles.

Purpose: Anecdotal reports have praised the benefits of cold exposure, exemplified by activities like winter swimming and cold water immersion. Cold exposure has garnered acclaim for its potential to confer benefits and potentially alleviate diabetes. We posited that systemic cold temperature (CT, 4-8°C) likely influences the organism's blood components through ambient temperature, prompting our investigation into the effects of chronic cold exposure on type 2 diabetic (T2DM) mice and our initial exploration of how cold exposure mitigates the incidence of T2DM. Methods: The effects of CT (4-8°C) or room temperature (RT, 22-25°C) on T2DM mice were investigated. Mice blood and organ specimens were collected for fully automated biochemical testing, ELISA, HE staining, immunohistochemistry, and immunofluorescence. Glucose uptake was assessed using flow cytometry with 2-NBDG. Changes in potential signaling pathways such as protein kinase B (AKT), phosphorylated AKT (p-AKT), insulin receptor substrates 1 (IRS1), and phosphorylated IRS1 (p-IRS1) were evaluated by Western blot. Results: CT or CT mice plasma-derived extracellular vesicles (CT-EVs) remarkably reduced blood glucose levels and improved insulin sensitivity in T2DM mice. This treatment enhanced glucose metabolism, systemic insulin sensitivity, and insulin secretion function while promoting glycogen accumulation in the liver and muscle. Additionally, CT-EVs treatment protected against the streptozocin (STZ)-induced destruction of islets in T2DM mice by inhibiting ß-cell apoptosis. CT-EVs also shielded islets from destruction and increased the expression of p-IRS1 and p-AKT in adipocytes and hepatocytes. In vitro experiments further confirmed its pro-insulin sensitivity effect. Conclusion: Our data indicate that cold exposure may have a potentially beneficial effect on the development of T2DM, mainly through the anti-diabetic effect of plasma-derived EVs released during cold stimulation. This phenomenon could significantly contribute to understanding the lower prevalence of diabetes in colder regions.

Solitary Giant Primary Gastric Plasmacytoma Mimicking Gastric Adenocarcinoma In Situ.

A solitary extraosseous plasmacytoma is a rare type of plasma cell neoplasm. Its occurrence in the stomach is particularly unusual and can easily be mistaken for more common types of tumors. We describe a case involving a solitary extraosseous plasmacytoma in a patient who experienced weight loss as the sole symptom. Initially, the condition was misdiagnosed as gastric adenocarcinoma based on endoscopic biopsy results, leading to a gastrectomy after neoadjuvant chemotherapy. Subsequent examination of the pathological specimen revealed the presence of plasma cell neoplasia alongside a gastric adenocarcinoma in situ.

The Paradox of Hyperkalaemia: When Treatment Isn't the Answer.

Hyperkalaemia is a relatively common medical emergency that necessitates prompt and urgent intervention. There is an ongoing debate over the precise threshold for treating hyperkalaemia due to variability in clinical scenarios. This case report highlights the need to differentiate true hyperkalaemia from pseudohyperkalaemia by analysing serum and plasma potassium levels, thus avoiding unnecessary treatment and the risk of iatrogenic hypokalemia. This case report discusses an 89-year-old male who presented with recurrent falls and fluctuating serum potassium levels but showed no symptoms of hyperkalaemia and had no relevant drug history. Further investigation revealed an underlying myeloproliferative neoplasm with thrombocytosis, leading to the diagnosis of pseudohyperkalaemia, reflected by a significant discrepancy between serum and plasma potassium levels, showcasing the importance of considering pseudohyperkalaemia in patients with haematological malignancies and thrombocytosis.

Systematic literature review of published cases of reactive plasmacytosis in peripheral blood and bone marrow.

AIMS: This study aims to summarise published cases of reactive plasmacytosis to provide a resource to aid haematopathologists and clinicians in the diagnostic workup of reactive plasmacytosis. METHODS: We searched published articles on reactive plasmacytosis on PubMed, Scopus and Google Scholar. Data were screened following Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines. Cases were classified into six categories, namely: (1) infection, (2) angioimmunoblastic T-cell lymphoma (AITL), (3) other malignancies, (4) drug associated, (5) autoimmune diseases and (6) others. Plasma cell percentage in peripheral blood and/or bone marrow was tabulated. Descriptive statistics were reported as median with IQR, using JASP Team. RESULTS: 87 articles which reported on 146 patients were included. Infectious diseases represented most cases associated with reactive plasmacytosis (n=46, 31% of all cases), with viral infections being the most frequent (n=31, 21% of all cases). AITL was the second most frequent aetiology (n=34, 23% of all cases), followed by medications (n=28, 19% of all cases), other malignancies (n=18, 12% of all cases), miscellaneous aetiologies (n=11, 7% of all cases) and autoimmune diseases (n=9, 6% of all cases). The absolute and relative levels of plasma cells in each diagnostic category showed marked variation and ranges largely overlapped between categories. CONCLUSIONS: In patients with an increase in the number and/or proportion of plasma cells in peripheral blood and/or bone marrow, clinical context and a broad differential diagnosis are necessary to direct further evaluation and arrive at a correct diagnosis. Our literature review suggests that evaluation for infectious causes and AITL may be of the greatest yield in many cases.

Efficacy of intra-articular injection of combined platelet-rich-plasma (PRP) and hyaluronic acid (HA) in knee degenerative joint disease: a prospective, randomized, double-blind clinical trial.

PURPOSE: Intra-articular injections of hyaluronic acid (HA) and platelet-rich plasma (PRP) represent promising options in the conservative treatment of early stages of knee osteoarthritis (OA). Although the combined use of these two compounds seems to have a synergistic effect in pre-clinical studies, few clinical trials compared the association of PRP and HA with PRP and HA alone. The aim of study is to evaluate the efficacy of combined intra-articular injections of PRP and HA in the treatment of mild to moderate knee OA, compared to PRP and HA alone. METHODS: One hundred and seventy-four patients were prospectively enrolled and randomized to undergo three intra-articular injections of either HA or PRP or the combination of PRP and HA with a two-week interval period. Patients were evaluated before treatment and after 3, 6, and 12 months using Western Ontario and McMaster Universities Osteoarthritis (WOMAC) score, Knee Injury and Osteoarthritis Outcome Score, International Knee Documentation Committee subjective score, Visual Analogue Scale and TEGNER Activity Scores. RESULTS: All the treatments proved to be effective in reducing pain and improving joint function. The analysis of covariance did not show statistically significant differences among the three groups for any of the investigated outcome parameters after 6 and 12 months (WOMAC: p = 0.45 and p = 0.64, respectively). No significant differences were found in terms of adverse events (p = 0.49) and painkiller use (p = 0.28 and p = 0.56, respectively) among the three groups. CONCLUSION: This multicenter, prospective, randomized, double-blind controlled trial revealed that intra-articular injections of PRP + HA, PRP, or HA represent a safe and viable treatment with comparable efficacy in terms of pain relief and functional outcomes in mild to moderate knee OA.

Comparative analysis of microwave radiation generated by the interaction between electron beam and plasma under different methods.

This article presents a physical model that describes the interaction between surface electron beams and plasma. The dispersion relations for beam plasma interactions were derived using perturbation method and field matching methods. The study investigates how different parameters affect radiation frequency and bandwidth. The results indicate that as electron beam velocity increases, the associated kinetic energy also rises, leading to an increase in both the maximum radiation frequency and bandwidth at high frequencies. Conversely, the radiation bandwidth at low frequencies decreases. Similarly, a higher plasma density results in a greater maximum radiation frequency, but the high-frequency bandwidth decreases, while the low-frequency bandwidth increases. Additionally, when the electron density and electron velocity of the electron beam remain constant, increasing the plasma density can increase the microwave radiation frequency However, there exists a plasma density threshold, beyond which high-frequency electromagnetic waves are no longer radiated.

The effect of plasma activated water on antimicrobial activity of silver nanoparticles biosynthesized by cyanobacterium Alborzia kermanshahica.

BACKGROUND: Silver nanoparticles are extensively researched for their antimicrobial properties. Cold atmospheric plasma, containing reactive oxygen and nitrogen species, is increasingly used for disinfecting microbes, wound healing, and cancer treatment. Therefore, this study examined the effect of water activated by dielectric barrier discharge (DBD) plasma and gliding arc discharge plasma on the antimicrobial activity of silver nanoparticles from Alborzia kermanshahica. METHODS: Silver nanoparticles were synthesized using the boiling method, as well as biomass from Alborzia kermanshahica extract grown in water activated by DBD and GA plasma. The physicochemical properties of the synthesized nanoparticles were evaluated using UV-vis spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), zeta potential analysis, transmission electron microscopy (TEM), and gas chromatography-mass spectrometry (GC-MS) analysis. Additionally, the disk diffusion method was used to assess the antimicrobial efficacy of the manufactured nanoparticles against both Gram-positive and Gram-negative bacteria. RESULTS: The spectroscopy results verified the presence of silver nanoparticles, indicating their biosynthesis. The highest amount of absorption (1.049) belonged to the nanoparticles synthesized by boiling under GA plasma conditions. Comparing the FTIR spectra of the plasma-treated samples with DBD and GA revealed that the DBD-treated samples had more intense peaks, indicating that the DBD method proved to be more effective in enhancing the functional groups on the silver nanoparticles. The DLS results revealed that the boiling method synthesized silver nanoparticles under DBD plasma treatment had a smaller particle size (149.89 nm) with a PDI of 0.251 compared to the GA method, and the DBD method produced nanoparticles with a higher zeta potential (27.7 mV) than the GA method, indicating greater stability of the biosynthesized nanoparticles. Moreover, the highest antimicrobial properties against E. coli (14.333 ± 0.47 mm) were found in the DBD-treated nanoparticles. TEM tests confirmed that spherical nanoparticles attacked the E. coli bacterial membrane, causing cell membrane destruction and cell death. The GC-MS results showed that compounds like 2-methylfuran, 3-methylbutanal, 2-methylbutanal, 3-hydroxy-2-butanone, benzaldehyde, 2-phenylethanol, and 3-octen-2-ol were much higher in the samples that were treated with DBD compared to the samples that were treated with GA plasma. CONCLUSION: The research indicated that DBD plasma was more efficient than GA plasma in boosting the antimicrobial characteristics of nanoparticles. These results might be a cornerstone for future advancements in utilizing cold plasma to create nanoparticles with enhanced antimicrobial properties.

Chemical Exposomics in Human Plasma by Lipid Removal and Large-Volume Injection Gas Chromatography-High-Resolution Mass Spectrometry.

For comprehensive chemical exposomics in blood, analytical workflows are evolving through advances in sample preparation and instrumental methods. We hypothesized that gas chromatography-high-resolution mass spectrometry (GC-HRMS) workflows could be enhanced by minimizing lipid coextractives, thereby enabling larger injection volumes and lower matrix interference for improved target sensitivity and nontarget molecular discovery. A simple protocol was developed for small plasma volumes (100-200 µL) by using isohexane (H) to extract supernatants of acetonitrile-plasma (A-P). The HA-P method was quantitative for a wide range of hydrophobic multiclass target analytes (i.e., log Kow > 3.0), and the extracts were free of major lipids, thereby enabling robust large-volume injections (LVIs; 25 µL) in long sequences (60-70 h, 70-80 injections) to a GC-Orbitrap HRMS. Without lipid removal, LVI was counterproductive because method sensitivity suffered from the abundant matrix signal, resulting in low ion injection times to the Orbitrap. The median method quantification limit was 0.09 ng/mL (range 0.005-4.83 ng/mL), and good accuracy was shown for a certified reference serum. Applying the method to plasma from a Swedish cohort (n = 32; 100 µL), 51 of 103 target analytes were detected. Simultaneous nontarget analysis resulted in 112 structural annotations (12.8% annotation rate), and Level 1 identification was achieved for 7 of 8 substances in follow-up confirmations. The HA-P method is potentially scalable for application in cohort studies and is also compatible with many liquid-chromatography-based exposomics workflows.

Regenerative treatment of canine osteogenic lesions with Platelet-Rich Plasma and hydroxyapatite: a case report.

Introduction: This study examined the efficacy of a therapy based on a combination of Platelet Rich Plasma and hydroxyapatite nanoparticles in a severe clinical case involving a young Rottweiler with a complex spiral fracture of the tibia. Method: Following a worsening of the lesion after traditional surgical intervention, the subject was treated with the combined therapy. X-rays were taken at the following stages: immediately post-surgery, four weeks post-surgery, and 10 days post-treatment. Fracture gap and callus density measurements were obtained using ImageJ analysis, allowing for a detailed quantitative assessment of bone regeneration over time. Results: Post-operative radiographs indicated a clinical worsening of the fracture, revealing an increased fracture gap due to bone loss. However, significant improvements were observed ten days following the treatment, with a marked reduction in fracture gaps and increased callus density. These results demonstrated a notable acceleration in bone healing and callus formation compared to typical recovery times for similar lesions. Conclusion: The method showed potential for enhancing osteogenic regeneration, facilitating faster healing of serious orthopedic injuries compared to traditional methods.

Analyzing the Bioactivity of a Novel Bone Cement: An In Vitro Study.

AIM: To analyze the effect of bioactive bone cement (BBC) placed in a phosphate buffer saline solution in comparison to mineral trioxide aggregate (MTA). METHODOLOGY: Ten samples each of BBC (group 1) and MTA (group 2) were prepared and stored in a phosphate buffer saline solution. After three days of storage, white precipitates were formed on the surface of the samples. The solution with precipitates from each sample was analyzed for the presence of calcium and phosphate ions with coupled plasma atomic spectroscopy. RESULTS: BBC showed a significant amount of calcium and phosphate release after a seven-day storage period in phosphate buffer saline solution. Calcium release was significantly higher in group 1 (MTA) (p < 0.001) compared to that in group 2 (BBC), while group 2 (BBC) (p < 0.001) exhibited greater phosphate release compared to group 1 (MTA). CONCLUSION: BBC (group 2) retains its bioactivity when it comes into contact with a stimulated oral environment (STF). This demonstrates that BBC is bioactive in a simulated oral environment. Moreover, it retained good handling properties and could be easily manipulated into a dough form. Clinically, in cases of apical surgery, internal resorption or perforation repair where material placement poses difficulty, BBC will prove to be beneficial.

PRP improves the outcomes of autologous skin graft transplantation on the esophagus by promoting angiogenesis and inhibiting fibrosis and inflammation.

Background and Objectives: Autologous skin graft (ASG) transplantation is a challenging approach but a promising option for patients to prevent postoperative esophageal stricture. Nonetheless, the current strategies require improvement. We aimed to investigate the effectiveness of the injection of platelet-rich plasma (PRP) before skin graft transplantation for extensive esophageal defects after endoscopic resection. Methods: Standardized complete circular endoscopic resection (5 cm in length) was performed in 27 pigs allocated into 3 groups. The artificial ulcers were treated with a fully covered esophageal stent (control group), ASG (ASG group), and submucosal injection of PRP with ASG (PRP-ASG group). Macroscopic evaluation and histological analysis of the remolded esophagus were performed 7, 14, and 28 days after surgery. Results: The macroscopic evaluation indicated that submucosal injection of PRP before transplantation effectively promoted the survival rate of skin grafts and decreased the rate of mucosal contraction compared with those treated with ASG or stent alone. Histological analysis of submucosal tissue showed that this modified strategy significantly promoted wound healing of reconstructed tissues by enhancing angiogenesis, facilitating collagen deposition, and decreasing inflammation and fibrogenesis. Conclusions: These findings suggested that PRP might be used as a biological supplement to increase the esophageal skin graft survival rate and improve submucosal tissue remolding in a clinically relevant porcine model. With extremely low mucosal contraction, this novel combination strategy showed the potential to effectively prevent stenosis in extensive esophageal ulcers.

Differences in survival of patients with multiple myeloma in rural versus metropolitan regions: Analysis of population data of an Australian local health district.

OBJECTIVE: The objective of this study is to determine if there are differences in outcome for patients diagnosed with multiple myeloma in a rural setting compared to a metropolitan setting and which factors influence these outcomes. DESIGN: Retrospective cohort study. SETTING: Illawarra Shoalhaven Local Health District. PARTICIPANTS: A total of 391 patients diagnosed with multiple myeloma between 2000 and 2022. MAIN OUTCOME MEASURES: Treatment and survival outcomes of these patients. RESULTS: Patients being treated in a rural cancer care centre had lower overall survival compared to those treated at a metropolitan cancer care centre (median OS = 44.4 months vs. 80.2 months, p = 0.002), despite access to similar treatments by the same group of haematologists. There was a significantly higher rate of upfront autologous transplantation (38% vs. 20%, p = 0.001) and higher rate of inclusion in clinical trials (16% vs. 7%, p = 0.021) in patients treated at a metropolitan cancer care centre compared to the rural cancer care centre. CONCLUSIONS: Multiple myeloma patients treated at a rural centre had shorter survival compared to patients treated at a metropolitan centre, and this may be related to lower rates of autologous transplantation and inclusion in clinical trials.

Photo-Amplified Plasma Membrane Rupture by Membrane-Anchoring NIR-II Small Molecule Design for Improved Cancer Photoimmunotherapy.

Immunotherapy is a promising cancer treatment method for eradicating tumor cells by enhancing the immune response. However, there are several major obstacles to conventional phototherapy-mediated immune responses, including inadequate immunogenicity and immunosuppressive environment. Here, we present a novel photoimmunotherapy modality-the development of membrane-anchoring small molecule inducing plasma membrane rupture (PMR) by NIR-II photo-stimulation, thus evoking cell necrotic death and enhancing antitumor immunotherapy. Our top-performing membrane-anchoring small molecule (CBT-3) exhibits temperature-tunable PMR efficiency, allowing rapid necrotic death in cancer cells at 50 µM dose by using exogenous NIR-II light-mediated mild photothermal effect (1064 nm, 0.6 W cm-2). Further evidence indicated that this gentle therapeutic approach activated inflammatory signaling pathways in cells, enhanced immunogenic cell death, and reshaped the immunosuppressive tumor microenvironment, ultimately promoting systemic antitumor immune responses in vivo. This study represents the first instance of utilizing NIR-II photo-amplified PMR effect based on membrane-anchoring small molecule, providing a novel avenue for advancing cancer photoimmunotherapy.

Proteomic analysis of plasma-derived extracellular vesicles: pre- and postprandial comparisons.

Extracellular vesicles (EVs) are key in intercellular communication, carrying biomolecules like nucleic acids, lipids, and proteins. This study investigated postprandial characteristics and proteomic profiles of blood-derived EVs in healthy individuals. Twelve participants fasted overnight before baseline assessments. After consuming a controlled isocaloric meal, EVs were isolated for proteomic and flow cytometric analysis. Plasma triacylglyceride levels confirmed fasting completion, while protein concentrations in plasma and EVs were monitored for postprandial stability. Proteomic analysis identified upregulated proteins related to transport mechanisms and epithelial/endothelial functions postprandially, indicating potential roles in physiological responses to nutritional intake. Enrichment analyses revealed vesicle-related pathways and immune system processes. Flow cytometry showed increased expression of CD324 on CD9+CD63+CD81+ large extracellular vesicles postprandially, suggesting an epithelial origin. These findings offer valuable insights into postprandial EV dynamics and their potential physiological significance, highlighting the need for stringent fasting guidelines in EV studies to account for postprandial effects on EV composition and function.

Epilipidomics reveals lipid fatty acid and headgroup modification in gas plasma-oxidized biomembranes.

Lipids, possessing unsaturated fatty acid chains and polar regions with nucleophilic heteroatoms, represent suitable oxidation targets for autologous and heterologous reactive species. Lipid peroxidation products (LPPs) are highly heterogeneous, including hydroperoxides, alkenals, chlorination, or glycation. Accordingly, delineation of lipid targets, species type, resulting products, and oxidation level remains challenging. To this end, liposomal biomimetic models incorporating a phosphatidylcholine, -ethanolamine, and a sphingomyelin were used to deconvolute effects on a single lipid scale to predict potential modification product outcomes. To introduce oxidative modifications, gas plasma technology, a powerful pro-oxidant tool to promote LPP formation by forming highly abundant reactive species in the gas and liquid phases, was employed to liposomes. The plasma parameters (gas type/combination) were modified to modulate the resulting species-profile and LPP formation by enriching specific reactive species types over others. HR-LC-MS (Münzel and et al., 2017) [2] was employed for LPP identification. Moreover, the heavy oxygen isotope 18O was used to trace O2-incorporation into LPPs, providing first information on the plasma-mediated lipid peroxidation mechanism. We found that combination of lipid class and gas composition predetermined the type of attack: admixture of O2 to the plasma and the presence of nitrogen atoms with free electrons in the molecule lead to chlorination of the amide bond and headgroup. Here, atomic oxygen driven formation of hypochlorite is the major reactive species. In contrast, POPC yields mainly to LPPs with oxidation of the oleic acid tail, especially truncations, epoxidation, and hydroperoxide formation. Here, singlet oxygen is assumingly the major driver. 18O labelling revealed that gas phase derived reactive species are dominantly incorporated into the LPPs, supporting previous findings on gas-liquid interface chemistry. In summary, we here provided the first insights into gas plasma-mediated lipid peroxidation, which, employed in more complex cell and tissue models, may support identifying mechanisms of actions in plasma medicine.

Mechanism of action and impact of thiol homeostasis on efficacy of an enzyme replacement therapy for classical homocystinuria.

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is characterized by elevated plasma and tissue homocysteine levels. There is no cure, but HCU is typically managed by methionine/protein restriction and vitamin B6 supplementation. Enzyme replacement therapy (ERT) based on human CBS has been developed and has shown significant efficacy correcting HCU phenotype in several mouse models by bringing plasma total homocysteine below the clinically relevant 100 µM threshold. As the reactive nature of homocysteine promotes disulfide formation and protein binding, and ERT is unable to normalize plasma total homocysteine levels, the mechanism of action of ERT in HCU remains to be further characterized. Here we showed that only a reduced homocysteine serves as a substrate for CBS and its availability restricts the homocysteine-degrading capacity of CBS. We also demonstrated that cells export homocysteine in its reduced form, which is efficiently metabolized by CBS in the culture medium. Availability of serine, a CBS co-substrate, was not a limiting factor in our cell-based model. Biological reductants, such as N-acetylcysteine, MESNA or cysteamine, increased the availability of the reduced homocysteine and thus promoted its subsequent CBS-based elimination. In a transgenic I278T mouse model of HCU, administration of biological reductants significantly increased the proportion of protein-unbound homocysteine in plasma, which improved the efficacy of the co-administered CBS-based ERT, as evidenced by significantly lower plasma total homocysteine levels. These results clarify the mechanism of action of CBS-based ERT and unveil novel pharmacological approaches to further increase its efficacy.