Search Results (351)

Background: With the increasing shift in drug design away from classical drug targets towards the modulation of protein-protein interactions, synthetic peptides are gaining increasing relevance. The synthesis and purification of peptides via solid-phase peptide synthesis (SPPS) strongly rely on trifluoroacetic acid (TFA) as a cleavage agent and ion-pairing reagent, respectively, resulting in peptides being obtained as TFA salts. Although TFA has excellent properties for peptide production, numerous studies highlight the negative impact of using peptides from TFA⁻ salts in biological assays. Methods: Investigated peptides were synthesized via SPPS and the TFA⁻ counterion was exchanged for Cl⁻ via freeze-drying in different concentrations of HCl. Detection and quantification of residual TFA⁻ were carried out via FT-IR, ¹⁹F-NMR, and HPLC using an evaporative light-scattering detector (ELSD). A liposomal fluorescence assay was used to test for the influence of the counterion on the peptides’ passive membrane permeability. Results: All TFA⁻ detection methods were successfully validated according to ICH guidelines. TFA⁻ removal with 10 mM HCl was determined to be the optimal condition. No impact on peptide purity was observed at all HCl concentrations. Influences on permeability coefficients depending on peptide sequence and salt form were found. Conclusions: This study presents a systematic investigation of the removal of TFA⁻ counterions from synthetic peptides and their replacement with Cl⁻ counterions. Detected counterion contents were used to understand the impact of sequence differences, especially positive charges, on the amount and potential localization of counterions. Our findings emphasize the importance of counterion quantification and specification in assays with synthetic peptides. Full article

Alperujo, a solid by-product from the two-phase olive oil extraction process, poses significant environmental challenges due to its high organic load, phytotoxicity, and phenolic content. At the same time, it represents a promising feedstock for recovering value-added compounds such as phenols and volatile fatty acids (VFAs). When used as a substrate for white rot fungi (WRF), it also produces ligninolytic enzymes. This study explores the use of two native WRF, Anthracophyllum discolor and Stereum hirsutum, for the biotransformation of alperujo under solid-state fermentation conditions, with and without supplementation of copper and manganese, two cofactors known to enhance fungal enzymatic activity. S. hirsutum stood out for its ability to release high concentrations of phenolic compounds (up to 6001 ± 236 mg gallic acid eq L⁻¹) and VFAs (up to 1627 ± 325 mg L⁻¹) into the aqueous extract, particularly with metal supplementation. In contrast, A. discolor was more effective in degrading phenolic compounds within the solid matrix, achieving a 41% reduction over a 30-day period. However, its ability to accumulate phenolics and VFAs in the extract was limited. Both WRF exhibited increased enzymatic activities (particularly Laccase and Manganese Peroxidase) with the addition of Cu-Mn, highlighting the potential of the aqueous extract as a natural source of biocatalysts. Phytotoxicity assays using Solanum lycopersicum seeds confirmed a partial detoxification of the treated alperujo. However, none of the fungi could entirely eliminate inhibitory effects on their own, suggesting the need for complementary stabilization steps before agricultural reuse. Overall, the results indicate that S. hirsutum, especially when combined with metal supplementation, is better suited for valorizing alperujo through the recovery of bioactive compounds. Meanwhile, A. discolor may be more suitable for detoxifying the solid phase strategies. These findings support the integration of fungal pretreatment into biorefinery schemes that valorize agroindustrial residues while mitigating environmental issues. Full article

Lifileucel, a tumor-infiltrating lymphocyte (TIL) cell therapy approved for advanced melanoma, demonstrates promise for treating other solid tumors, including endometrial cancer (EC). The current study evaluates the feasibility of manufacturing TILs from EC tumors using Iovance’s proprietary 22-day Gen2 manufacturing process. Key parameters, including TIL yield, viability, immune phenotype, T-cell receptor clonality, and cytotoxic activity, were assessed. Of the 11 EC tumor samples processed at research scale, 10 (91%) successfully generated >1 × 10⁹ viable TIL cells, with a median yield of 1.1 × 10¹⁰ cells and a median viability of 82.8%. Of the four EC tumor samples processed at full scale, all achieved the pre-specified TVC and viability targets. Putative tumor-reactive T-cell clones were maintained throughout the manufacturing process. Functional reactivity was evidenced by the upregulation of 4-1BB in CD8+ T cells, OX40 in CD4+ T cells, and increased production of IFN-γ and TNF-α upon autologous tumor stimulation. Furthermore, antitumor activity was confirmed using an in vitro autologous tumor organoid killing assay. These findings demonstrate the feasibility of ex vivo TIL expansion from EC tumors. This study provides a rationale for the initiation of the phase II clinical trial IOV-END-201 (NCT06481592) to evaluate lifileucel in patients with advanced EC. Full article

Knowledge about the composition (volatile and non-volatile) and functionality of natural extracts from Mediterranean plants serves as a basis for their further application. In this study, five selected plants were used for the extraction of plant metabolites. Leaves and flowers of Critmum maritimum, Rosmarinus officinalis, Olea europea, Phylliera latifolia and Mellisa officinalis were collected, and a total of 12 extracts were prepared. Extractions were performed under microwave-assisted conditions, with two solvent types: water (W) and a hydroalcoholic (ethanolic) solution (HA). Detailed extract analysis was conducted. Phenolics were analyzed by detecting individual bioactive compounds using high-performance liquid chromatography and by calculating total phenolic and total flavonoid content through spectrophotometric analysis. Higher concentrations of total phenolics and total flavonoids were obtained in the hydroalcoholic extracts, with the significantly highest total phenolic and flavonoid values in the rosemary hydroalcoholic extract (3321.21 mg_GAE/L) and sea fennel flower extract (1794.63 mg_QE/L), respectively; and the lowest phenolics in the water extract of olive leaves (204.55 mg_GAE/L) and flavonoids in the water extracts of sea fennel leaves, rosemary, olive and mock privet (around 100 mg_QE/L). Volatile organic compounds (VOC) were detected using HS-SPME/GC–MS (Headspace Solid-Phase Microextraction coupled with Gas Chromatography-Mass Spectrometry), and antioxidant capacity was estimated using DPPH (2,2-diphenyl-1-picrylhydrazyl assay) and FRAP (Ferric Reducing Antioxidant Power) methods. HS-SPME/GC–MS analysis of samples revealed that sea fennel had more versatile profile, with the presence of 66 and 36 VOCs in W and HA sea fennel leaf extracts, 52 and 25 in W and HA sea fennel flower extracts, 57 in rosemary W and 40 in HA, 20 in olive leaf W and 9 in HA, 27 in W mock privet and 11 in HA, and 35 in lemon balm W and 10 in HA extract. The lowest values of chlorophyll a were observed in sea fennel leaves (2.52 mg/L) and rosemary (2.21 mg/L), and chlorophyll b was lowest in sea fennel leaf and flower (2.47 and 2.25 mg/L, respectively), while the highest was determined in olive (6.62 mg/L). Highest values for antioxidant activity, determined via the FRAP method, were obtained in the HA plant extracts (up to 11,216 mg_AAE/L for lemon balm), excluding the sea fennel leaf (2758 mg_AAE/L) and rosemary (2616 mg_AAE/L). Considering the application of these plants for fresh fish preservation, antimicrobial activity of water extracts was assessed against Vibrio fischeri JCM 18803, Vibrio alginolyticus 3050, Aeromonas hydrophila JCM 1027, Moraxella lacunata JCM 20914 and Yersinia ruckeri JCM 15110. No activity was observed against Y. ruckeri and P. aeruginosa, while the sea fennel leaf showed inhibition against V. fisheri (inhibition zone of 24 mm); sea fennel flower was active against M. lacunata (inhibition zone of 14.5 mm) and A. hydrophila (inhibition zone of 20 mm); and rosemary and lemon balm showed inhibition only against V. fisheri (inhibition zone from 18 to 30 mm). This study supports the preparation of natural extracts from Mediterranean plants using green technology, resulting in extracts rich in polyphenolics with strong antioxidant potential, but with no clear significant antimicrobial efficiency at the tested concentrations. Full article

Assessment for the presence or absence of lupus anticoagulant (LA) represents a common investigation in hemostasis laboratories. In particular, LA represents one of the laboratory criteria for the diagnosis of definite antiphospholipid syndrome (APS). The other laboratory criteria are the solid phase assays (anticardiolipin (aCL) and anti-β2Glycoprotein I (aβ2GPI) antibodies of IgG and IgM isotypes). Current International Society on Thrombosis and Haemostasis (ISTH) guidance recommends testing LA by at least two tests based on different principles, with the activated partial thromboplastin time (aPTT) and dilute Russell viper venom time (dRVVT) being preferred. Additional assays may be used in addition, or instead of these assays in particular situations. For example, aPTT and dRVVT assays are very sensitive to the presence of various anticoagulants, and this may lead to false-positive identification of LA. This is particularly problematic in the age of the DOACs (direct oral anticoagulants), which are now the leading anticoagulants in use worldwide. We review recent literature on LA testing as well as our local practice to provide an update on this common test procedure. Our experience should be useful for laboratories struggling with LA interpretation for diagnosis or exclusion of APS. Full article

The garden Nasturtium (Tropaeolum majus L.) is increasingly consumed worldwide due to its culinary appeal and perceived health benefits. However, the chemical markers underlying its functional properties remain insufficiently characterized. Building on evidence from a recent human pilot study confirming both high acceptability and dietary safety, we conducted a comprehensive volatilomic and phytochemical analysis of T. majus flowers and their juice. Headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC-MS) was employed to establish the volatilomic fingerprint of floral tissues and juice. Our analysis revealed a striking dominance of benzyl isothiocyanate and benzonitrile, which together accounted for 88% of the total volatile organic metabolites (VOMs) in the juice, 67% and 21%, respectively. In the floral tissues, benzyl isothiocyanate was even more prevalent, representing 95% of the total volatile profile. Complementary in vitro assays confirmed a substantial total phenolic content and strong antioxidant activity in the flowers. These findings provide a robust chemical rationale for the potential health-promoting attributes of T. majus, while identifying key volatilomic markers that could support future functional and safety claims. In parallel, a benefit–risk assessment framework is discussed in accordance with the European Food Safety Authority (EFSA) guidelines for the Qualified Presumption of Safety (QPS) of edible flowers. Given that both benzyl isothiocyanate and benzonitrile are classified as Cramer Class III substances, a conservative intake threshold of 1.5 μg/kg body weight per day is proposed. To enable quantitative exposure modeling and support the derivation of a tolerable daily intake (TDI), future studies should integrate organic solvent-based extraction methodologies to estimate the total volatile load per gram of floral biomass. This would align risk–benefit assessments with the EFSA’s evolving framework for novel foods and functional ingredients. Full article

Polyphenols are valuable contributors to skin health, offering potent antioxidant and anti-inflammatory effects that help counteract the process of inflammaging. According to the literature, Urtica dioica L. is a rich source of polyphenolic compounds, suggesting its potential for applications in cosmetology and dermatology. This study aimed to evaluate the antioxidant and anti-inflammatory activity of polyphenol-rich fractions isolated from U. dioica leaves (UdLs) and flowers (UdFs) using human skin cells subjected to oxidative stress and lipopolysaccharide (LPS) stimulation, respectively. Extracts were obtained via an accelerated solvent extraction and further purified by a solid-phase extraction to concentrate their polyphenolic content. Their chemical composition was analyzed using UPLC-DAD-MS. Biological activity was assessed through cytotoxicity assays (NR and MTT), chemical and cellular antioxidant assays (DPPH, ABTS, FRAP, CUPRAC, TPC, and H₂DCFDA), an evaluation of antioxidant enzyme activity (SOD, CAT), lipid peroxidation (MDA), and cytokine production (IL-1β, IL-6, IL-10). Our study showed that both fractions were abundant in phenolic compounds, with chlorogenic acid identified as the predominant constituent. UdLs contained higher levels of phenolic acids, whereas the UdF was richer in flavonoids, particularly derivatives of isorhamnetin. Both the UdL and UdF were non-cytotoxic and exhibited strong radical scavenging potential, with the UdL being slightly more effective. They significantly reduced intracellular ROS levels, enhanced the activity of antioxidant enzymes, and attenuated lipid peroxidation in cells exposed to oxidative stress. Moreover, both fractions reduced the secretion of pro-inflammatory cytokines in LPS and H₂O₂-stimulated fibroblasts. These results highlight the potential of polyphenolic fractions derived from U. dioica leaves and flowers as multifunctional ingredients for anti-aging and skin-protective cosmetics. Full article

Modern dermocosmetics combine the effectiveness of active substances with the benefits of percutaneous penetration enhancers to address skin issues such as hyperpigmentation. In this study, three bioactive tripeptides (with amino acid sequences CSF, CVL, and CSN) with previously confirmed tyrosinase inhibition activity were synthesized using the solid-phase synthesis method. The structures of the obtained peptides were determined. In addition, elastase in silico and in vitro inhibition assays were carried out. The tripeptides were subsequently encapsulated into liposomes, for which key physicochemical parameters were determined, including size, zeta potential, and encapsulation efficiency. The average diameter of the prepared liposomes was approximately 100 nm across all samples. The prepared carriers were found to be stable and exhibited no cytotoxicity toward reconstructed human epidermis cells. The peptides achieved an encapsulation efficiency of approximately 20–30%, with no significant differences observed between the cationic and anionic vesicles. Liposomes containing the CSF tripeptide, which showed the strongest tyrosinase-inhibiting effect, did not transport the peptide through the human skin in an ex vivo assay to permit quantification in the receptor solution, but facilitated penetration and retention of the tripeptide within the epidermis (4.65 ± 1.81 μg/cm²). These findings suggest that the prepared liposomes may serve as valuable carriers of bioactive tripeptides in anti-aging cosmetics. Full article

Heavy metal and cyanide contamination in soil presents serious environmental and ecological concerns due to their persistence, bioavailability, and toxicity to soil biota. In this study, a novel solid-phase direct contact bioassay kit was developed using immobilized Chlorella vulgaris spheres to evaluate the toxicity of soils contaminated with mercury (Hg²⁺), silver (Ag⁺), copper (Cu²⁺), and cyanide (CN⁻). The assay was designed using 25 mL glass vials in which algal spheres were directly exposed to spiked soils for 72 h without the need for pollutant extraction. Oxygen evolution in the headspace was measured as the primary endpoint, alongside optical density and chlorophyll a fluorescence (OJIP) to assess photosynthetic inhibition. The assay demonstrated high sensitivity and reproducibility, with strong correlations (R² > 0.93) between oxygen evolution and optical density. EC₅₀ values based on oxygen evolution were 4.43, 4.18, 3.10, and 61.3 mg/kg for Hg²⁺, Ag⁺, CN⁻, and Cu²⁺, respectively, and 7.8, 7.4, 2.9, and 29.7 mg/kg based on optical density. The relatively higher EC₅₀ for copper was attributed to its biological role as an essential micronutrient. OJIP transient profiles supported the observed photosynthetic inhibition, particularly under Hg²⁺, Ag⁺, and CN⁻ exposure. The present study overcomes the limitations of conventional chemical analyses by providing a rapid, low-cost, and ecologically relevant tool for direct soil toxicity assessment, with potential applications in environmental monitoring and contaminated site evaluation. Full article

The quality of persimmon wine is closely related to various compounds, including polysaccharides. Polysaccharides are an essential class of macromolecules that modulate the wine’s chemical and physical characteristics by influencing the colloidal state or interacting with other compounds through non-covalent bonds. Polyphenols, on the other hand, exhibit antioxidant properties and effectively neutralize free radicals. This study employed Luotian sweet persimmons and Brassica napus (rapeseed) as core ingredients for producing functional fermented wine. Using GC-MS, rapeseed polysaccharides were subjected to trifluoroacetic acid hydrolysis and then derivatized via silylation for qualitative analysis of their monosaccharide composition. Molecular docking and molecular dynamics simulations were performed to provide molecular-level insights into the interactions between D-glucopyranose from rapeseed polysaccharides and quercetin, a polyphenol present in persimmon wine. The objective was to explore the binding mechanisms of these compounds during fermentation and to assess how these molecular interactions in-fluence the wine’s flavor and stability. In addition, volatile flavor compounds in two types of persimmon wine (pure persimmon wine and oleoresin-enriched persimmon wine) were qualitatively and quantitatively analyzed using headspace solid-phase microextraction (SPME) combined with gas chromatography–mass spectrometry (GC-MS). The results reveal that D-glucopyranose forms hydrogen bonds with quercetin, modulating its redox behavior and thereby enhancing the antioxidant capacity of persimmon wine. The results from four in vitro antioxidant assays, including DPPH, ABTS, FRAP, and vitamin C analysis, demonstrate that the addition of rapeseed flowers improved the antioxidant activity of persimmon wine. HS-SPME-GC-MS analysis revealed that esters, alcohols, and aldehydes were the primary components contributing to the aroma of persimmon wine. Persimmon wines with varying levels of oleoresin addition exhibited significant differences in the contents of key compounds, which subsequently influenced the aroma complexity and flavor balance. In conclusion, these findings provide reliable data and a theoretical foundation for understanding the role of rapeseed flower in regulating the aroma profile of persimmon wine. These findings also offer theoretical support for a deeper understanding of the fermentation mechanisms of persimmon wine while providing practical guidance to optimize production processes, ultimately improving both product flavor and stability. This study fills a critical academic gap in understanding microscopic molecular interactions during fermentation and offers a novel perspective for innovation in the fermented food industry. Full article

Histatin 5 (Hst5) is an antifungal peptide (AFP) naturally produced by parotid glands with strong activity against Candida albicans. One of its mechanisms of action is the generation of reactive oxygen species (ROS) inside the C. albicans cells. Despite being an important peptide for the human innate immune response, its activity is reduced or inactivated by proteolytic degradation caused by salivary enzymes. To overcome this barrier, we used solid phase peptide synthesis (SPPS) to modify the Hst5 amino acid sequence improving its antifungal action and minimizing its degradation. We synthesized five peptides, three of which were based on the Hst5 functional domain. We determined that the smallest peptides (8WH5, 7WH5 and 6WH5) demonstrated the greatest antifungal action against C. albicans, including one fluconazole-resistant strain. Besides that, cationic-PAGE and HPLC assays showed that the degradation in saliva was slower for the smaller peptides than for 0WHst5 and WP113. Furthermore, 8WH5, 7WH5 and 6WH5 were found in the samples even after 8 h in whole saliva, while 0WHst5 and WP113 completely disappear after 1.5 h. Finally, we found that the smaller peptides were less fragmented than the 0WHst5 and WP113, so they were the smallest fragments of Hst5 to preserve its antifungal action with reduced degradation in whole saliva. Thus, they can be considered promising molecules for the treatment of C. albicans in the oral cavity. Full article

Amyloid beta (Aβ42 and Aβ40) aggregation, along with neurofibrillary tangles, is one of the major neurotoxic events responsible for the onset of Alzheimer’s disease. Many potent peptide-based inhibitors mainly focusing on central hydrophobic core Aβ16–20 (KLVFF) have been reported in recent years. Herein, we report pentapeptides 1–4, based on the β-turn-inducing fragment Aβ19–23 (FFAED). The synthesis of peptides 1–4 was carried out using Fmoc/tBu-based solid-phase peptide synthesis technique, and it was found that pentapeptide 3 potently inhibit the aggregation propensity of Aβ42, when incubated with it at 37 °C for 48 h. The aggregation inhibition study was conducted using thioflavin T-based fluorescence assay and circular dichroism spectroscopy, and supported by transmission electron microscope imaging. The conformational change on the aggregation of Aβ42 and aggregation inhibition by peptides 1–4 was further evaluated using ¹H–¹⁵N HSQC NMR spectroscopy. The results demonstrated that the most potent analog, peptide 3, effectively disrupts the aggregation process. This study is the first to demonstrate that an Aβ19–23 fragment mimic can disrupt the aggregation propensity of Aβ42. Full article

Reservoirs and downstream rivers draining Oregon’s Cascade Range provide critical water supplies for over 1.5 million residents in dozens of communities. These waters also support planktonic and benthic cyanobacteria that produce cyanotoxins that may degrade water quality for drinking, recreation, aquatic life, and other beneficial uses. This 2016–2020 survey examined the sources and transport of four cyanotoxins—microcystins, cylindrospermopsins, anatoxins, and saxitoxins—in six river systems feeding 18 drinking water treatment plants (DWTPs) in northwestern Oregon. Benthic cyanobacteria, plankton net tows, and (or) Solid-Phase Adsorption Toxin Tracking (SPATT) samples were collected from 65 sites, including tributaries, reservoirs, main stems, and sites at or upstream from DWTPs. Concentrated extracts (320 samples) were analyzed with enzyme-linked immuno-sorbent assays (ELISA), resulting in >90% detection. Benthic cyanobacteria (n = 80) mostly Nostoc, Phormidium, Microcoleus, and Oscillatoria, yielded microcystins (76% detection), cylindrospermopsins (41%), anatoxins (45%), and saxitoxins (39%). Plankton net tow samples from tributaries and main stems (n = 94) contained saxitoxins (84%), microcystins (77%), anatoxins (25%), and cylindrospermopsins (22%), revealing their transport in seston. SPATT sampler extracts (n = 146) yielded anatoxins (81%), microcystins (66%), saxitoxins (37%), and cylindrospermopsins (32%), indicating their presence dissolved in the water. Reservoir plankton net tow samples (n = 15), most often containing Dolichospermum, yielded microcystins (87%), cylindrospermopsins (73%), and anatoxins (47%), but no saxitoxins. The high detection frequencies of cyanotoxins at sites upstream from DWTP intakes, and at sites popular for recreation, where salmon and steelhead continue to exist, highlight the need for additional study on these cyanobacteria and the factors that promote production of cyanotoxins to minimize effects on humans, aquatic ecosystems, and economies. Full article

Culinary herbs and spices are valued worldwide for their flavor, aroma, and medicinal benefits. They encompass diverse bioactive metabolites, such as polyphenols and terpenoids, which contribute to plant defense and offer anticarcinogenic, anti-inflammatory, antioxidant, and cognitive-enhancing effects. This study aimed to establish the volatile fingerprint of culinary herbs (lemon verbena, chives, basil, sage, coriander, and parsley) and spices (curcuma, nutmeg, cumin, black pepper, Jamaica pepper, and juniper berry) using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME/GC-MS). The predominant volatile organic metabolites (VOMs) identified were subjected to in silico molecular docking simulations of anti-Alzheimer’s (e.g., acetylcholinesterase (AChE), butyrylcholinesterase (BChE)), antioxidants (e.g., monoamine oxidase B (MAO-B), inducible nitric oxide synthase (iNOS)), and anti-inflammatory receptors (e.g., 5-lipoxygenase (5-LOX), cyclooxygenase-2 (COX-2)). The culinary herb and spice extracts were also subjected to in vitro assays to evaluate their potential as antioxidant (DPPH, ABTS, and ORAC) and anti-inflammatory (% protein denaturation) agents. A total of 121 VOMs were identified in the culinary herbs and spices, with the predominant chemical families being monoterpenoids (48.3%), sesquiterpenoids (14.0%), esters (11.9%), and carbonyl compounds (8.8%). In silico molecular docking simulations revealed that cuminaldehyde, β-caryophyllene, γ-curcumene, germacrene D, and τ-cadinol exhibited the strongest inhibitory activities against the selected receptors. Among the extracts, Jamaica pepper showed the highest antioxidant and anti-inflammatory activities, while lemon verbena exhibited the lowest ones. These findings highlight the promising potential of the studied culinary herbs and spices in the modulation of inflammatory processes related to Alzheimer’s disease. However, further investigations, particularly clinical studies, are recommended to validate these results and explore their therapeutic applications. Full article

Background/Objectives: According to recent guidelines, including the guideline on bioanalytical method validation issued by the European Medicine Agency, the stability of clinical analytes should be known. We summarize human α₁-proteinase inhibitor (A1PI) and urea stability data in relevant matrices, as these analytes are usually measured in clinical A1PI studies. Methods: Stability samples with appropriate A1PI concentrations were prepared in a citrated human reference plasma pool and a matrix mimicking bronchoalveolar lavage (BAL) solution. These samples were kept at −20 °C and −60 °C for up to 24 months. A1PI protein was measured with a nephelometric method and an enzyme-linked immunosorbent assay using paired commercially available polyclonal antibodies. A1PI elastase inhibitory activity was determined with an elastase complex formation immunosorbent assay that combines A1PI complex formation with a solid phase-immobilized elastase and immunological detection of the formed A1PI-elastase complex and urea in samples kept at −20 °C only with an enzymatic assay. Results: Overall, the stability criterion (100 ± 20%) was met for the analytes A1PI protein and A1PI activity at both temperatures during storage of BAL-mimicking and plasma samples for 15 and 24 months, respectively; urea was stable in both matrices at −20 °C for 18 months. Plasma samples showed smaller drops in concentration over storage time than BAL-mimicking samples. As expected, the reduction of A1PI elastase inhibitory activity was more pronounced than that of A1PI protein. Interestingly, the analyte concentration did not significantly influence the results in either of the sample matrices. Conclusions: The data confirmed the appropriate stability of the three analytes in the matrices of citrated plasma and BAL-mimicking samples for at least up to 15 months. Full article