Constructing network structures to enhance stability and target deposition of selenium nanoparticles via amphiphilic sodium alginate and alkyl glycosides.

Dietary selenium (Se) supplementation has recently received increasing attention; however, Selenium nanoparticles (SeNPs) exhibit poor stability and tend to aggregate in aqueous solution. Therefore, enhancing the stability of SeNPs and their effective delivery to plants remain challenging. In this study, sodium alginate (SA) and lysozyme (LZ) were reacted via the wet-heat Maillard reaction (MR) to obtain amphiphilic alginate-based polymers (SA-LZ). Alkyl glycosides (APG) were introduced into SA-LZ to enhance the deposition of SeNPs in leaves. Thus, a renewable and degradable polysaccharide-based material (SA-LZ/APG) loaded with Se formed an amphiphilic alginate-based-based shell with a Se core. Notably, the encapsulation of SeNPs into a polysaccharide base (SA-LZ/APG) increased the stabilization of SeNPs and resulted in orange-red, zero-valent, monoclinic and spherical SeNPs with a mean diameter of approximately 43.0 nm. In addition, SA-LZ/APG-SeNPs reduced the interfacial tension of plant leaves and increased the Se content of plants compared to the blank group. In vitro studies have reported that SA-LZ/APG-SeNPs and SA-LZ-SeNPs have significantly better clearance of DDPH and ABTS than that of APG-SeNPs. Thus, we believe that SA-LZ/APG is a promising smart delivery system that can synergistically enhance the stability of SeNPs in aqueous solutions and improve the bioavailability of Se nutrient solutions.

Design and synthesis of novel carbohydrate-amino acid hybrids and their antioxidant and anti-ß-amyloid aggregation activity.

Herein we report the synthesis of new furanoid sugar amino acids and thioureas, prepared by coupling aromatic amino acids and dipeptides with isothiocyanato- functionalized ribofuranose ring. Since carbohydrate-derived structures possess many biological activities, synthesized compounds were evaluated as anti-amyloid and antioxidant agents. The anti-amyloid activity of the studied compounds was evaluated based on their potential to destroy amyloid fibrils of intrinsically disordered Aß40 peptide and globular hen egg-white (HEW) lysozyme. The destructive efficiency of the compounds differed between the studied peptides. While the destruction activity of the compounds on the HEW lysozyme amyloid fibrils was negligible, the effect on Aß40 amyloid fibrils was significantly higher. Furanoid sugar α-amino acid 1 and its dipeptide derivatives 8 (Trp-Trp) and 11 (Trp-Tyr) were the most potent anti-Aß fibrils compounds. The antioxidant properties of synthesized compounds were estimated by three complementary in vitro assays (DPPH, ABTS, and FRAP). The ABTS assay was the most sensitive for assessing the radical scavenging activity of all tested compounds compared to the DPPH test. Significant antioxidant activity was detected for compounds in the group of aromatic amino acids depending on the present amino acid, with the highest activity in the case of dipeptides 11 and 12 containing the Tyr and Trp moiety. Regarding the FRAP assay, the best reducing antioxidant potential revealed Trp-containing compounds 5, 10, and 12.

Green tea leaf constituents inhibit the formation of lysozyme amyloid aggregates: An effect of mutual interactions.

Amyloidoses represent a group of pathological conditions characterized by amyloid fibrils accumulating in the form of deposits in intra- or extracellular space, leading to tissue damage. The lysozyme from hen egg-white (HEWL) is often used as a universal model protein to study the anti-amyloid effects of small molecules. The in vitro anti-amyloid activity and mutual interactions of green tea leaf constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF) and their equimolar mixtures were studied. The inhibition of HEWL amyloid aggregation was monitored by a Thioflavin T fluorescence assay and atomic force microscopy (AFM). The interactions of the analyzed molecules with HEWL were interpreted by ATR-FTIR and protein-small ligand docking studies. EGCG was the only substance efficiently inhibiting amyloid formation (IC50 âˆ¼193 µM), slowing the aggregation process, reducing the number of fibrils and partially stabilizing the secondary structure of HEWL. Compared to EGCG alone, EGCG-containing mixtures displayed lower overall anti-amyloid efficacy. The decrease in efficiency results from (a) the spatial interference of GA, CF and EC with EGCG while binding to HEWL, (b) the propensity of CF to form a less active adduct with EGCG, which participates in interactions with HEWL in parallel with pure EGCG. This study confirms the importance of interaction studies, revealing the possible antagonistic behavior of molecules when combined.

Structural insights into thrombolytic activity of destabilase from medicinal leech.

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 µs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure-activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.

Milk Proteins-Their Biological Activities and Use in Cosmetics and Dermatology.

Milk and colostrum have high biological potential, and due to their natural origin and non-toxicity, they have many uses in cosmetics and dermatology. Research is ongoing on their potential application in other fields of medicine, but there are still few results; most of the published ones are included in this review. These natural products are especially rich in proteins, such as casein, ß-lactoglobulin, α-lactalbumin, lactoferrin, immunoglobulins, lactoperoxidase, lysozyme, and growth factors, and possess various antibacterial, antifungal, antiviral, anticancer, antioxidant, immunomodulatory properties, etc. This review describes the physico-chemical properties of milk and colostrum proteins and the natural functions they perform in the body and compares their composition between animal species (cows, goats, and sheep). The milk- and colostrum-based products can be used in dietary supplementation and for performing immunomodulatory functions; they can enhance the effects of certain drugs and can have a lethal effect on pathogenic microorganisms. Milk products are widely used in the treatment of dermatological diseases for promoting the healing of chronic wounds, hastening tissue regeneration, and the treatment of acne vulgaris or plaque psoriasis. They are also increasingly regarded as active ingredients that can improve the condition of the skin by reducing the number of acne lesions and blackheads, regulating sebum secretion, ameliorating inflammatory changes as well as bestowing a range of moisturizing, protective, toning, smoothing, anti-irritation, whitening, soothing, and antiaging effects.

Synergistic Lysozyme-Photodynamic Therapy Against Resistant Bacteria based on an Intelligent Upconversion Nanoplatform.

The rapid emergence of drug-resistant bacteria has raised a great social concern together with the impetus for exploring advanced antibacterial ways. NIR-triggered antimicrobial photodynamic therapy (PDT) by lanthanide-doped upconversion nanoparticles (UCNP) as energy donor exhibits the advantages of high tissue penetration, broad antibacterial spectrum and less acquired resistance, but is still limited by its low efficacy. Now we designed a bio-inorganic nanohybrid and combined lysozyme (LYZ) with UCNP-PDT system to enhance the efficiency against resistant bacteria. Benefiting from the rapid adhesion to bacteria, intelligently bacteria-responsive LYZ release and synergistic LYZ-PDT effect, the nanoplatform achieves an exceptionally strong bactericidal capacity and conspicuous bacteriostasis on methicillin-resistant S. aureus. These findings pave the way for designing efficiently antibacterial nanomaterials and provide a new strategy for combating deep-tissue bacterial infection.

Inhibition behavior of Sennoside A and Sennoside C on amyloid fibrillation of human lysozyme and its possible mechanism.

Amyloid proteins were recognized as the crucial cause of many senile diseases. In this study, the inhibitory effects of Sennoside A (SA) and Sennoside C (SC) on amyloid fibrillation were evaluated by the combination of biophysical approaches and molecular docking tool using human lysozyme (HL) as amyloid-forming model. The results of thioflavin-T (ThT), 8-anilino-1-naphthalenesulfonic acid (ANS) and congo red (CR) assays indicated that both SA and SC could inhibit the amyloid fibrillation of HL in a dose-dependent manner. The IC50 value of SA and SC on HL fibrillation was 200.09 µM and 186.20 µM, respectively. These findings were further verified by transmission electron microscopy (TEM) and atomic force microscopy (AFM), which showed that the addition of SA or SC could sharply reduce the amyloid fibrillation of HL. Additionally, the interactions of HL with SA and SC were investigated by steady-state fluorescence spectra and molecular docking studies. The results suggested that both SA and SC could bind to the binding pocket of HL and form a stable complex mainly via hydrogen bonds, van-der-Waals forces and hydrophobic interactions. In conclusion, our experiments revealed that both SA and SC can significantly inhibit amyloid fibrillation of HL.

Synthesis and Characterization of Selenium Nanoparticles-Lysozyme Nanohybrid System with Synergistic Antibacterial Properties.

In the light of promising potency of selenium nanoparticles in biomedical applications, this is the first study to report the synergistic antibacterial activity of these nanoparticles and lysozyme. The nanohybrid system was prepared with various concentrations of each component. Resistance of Escherichia coli and Staphylococcus aureus was compared in the presence of individual Nano and Bio counterparts as well as the nanohybrid system. Upon interaction of SeNPs with Lysozyme, the nanohybrid system efficiently enhanced the antibacterial activity compared to the protein. Therefore, SeNPs play an important role in inhibition of bacterial growth at very low concentrations of protein; whereas very high amount of the protein is required to inhibit bacterial growth individually. On the other hand, lysozyme has also played a vital role in antibacterial property of SeNPs, inducing 100% inhibition at very low concentration of each component. Hence, presence of both nano and bio counterparts induced vital interplay in the Nanohybrid system. The aged samples also presented good stability of SeNPs both as the intact and complex form. Results of this effort highlight design of nanohybrid systems with synergistic antibacterial properties to overcome the emerging antibiotic resistance as well as to define fruitful applications in biomedicine and food safety.

Herbalome of Chandraprabha vati, a polyherbal formulation of Ayurveda prevents fibrillation of lysozyme by stabilizing aggregation-prone intermediate state.

Lysozyme amyloidosis (ALys) is caused by the deposition of amyloid-like fibrils of lysozyme in the tissues of the gastrointestinal tract, liver and kidneys. The treatment/prevention of ALys is not known yet. Therefore, searching for therapeutic agents for amyloidosis is of great value. In this study, we have examined the ability of the aqueous extract of herbalome (thirty herbal components) of Chandraprabha vati (EHCV), a polyherbal Ayurvedic formulation, to prevent fibrillation of lysozyme. Transmission electron microscopy and multiple biophysical techniques were used to examine the processes. We found complete inhibition of the fibrillation by EHCV, whereas none of the thirty ingredients of EHCV was able to prevent the reaction, solely. We also found the EHCV induced and stabilized secondary structures of aggregation-prone state (APS) of lysozyme. Moreover, an increase in the secondary structure and stability of APS were found to correlate with the inhibition reaction. We conclude that EHCV modulates the structure and stability of APS and converts it into an aggregation resistant state (ARS). We hypothesized that herbal components of Ayurvedic formulation may provide a combination of molecules, which could efficiently prevent aggregation reaction.

Comparison of Protein Precipitation Ability of Structurally Diverse Procyanidin-Rich Condensed Tannins in Two Buffer Systems.

The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[N-morpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined included Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5% galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages). and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF > LYS ≥ BSA. PP in GVS buffer was 1 to 2.25 times greater than that in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impacts mDP, galloylation, and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature at which GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.

Effect of particle functionalization and solution properties on the adsorption of bovine serum albumin and lysozyme onto silica nanoparticles.

Silica nanoparticles present an enormous potential as controlled drug delivery systems with high selectivity towards diseased cells. This application is directly related to the phenomenon of protein corona, characterized by the spontaneous adsorption of proteins on the nanoparticle surface, which is not fully understood. Here, we report an investigation on the influence of pH, ionic strength and temperature on the thermodynamics of interaction of bovine serum albumin protein (BSA) with non-functionalized silica nanoparticles (SiO2NPs). Complementary, we also investigated the ability of polyethylene glycol (PEG) and zwitterionic sulfobetaine (SBS) surface-modified nanoparticles to prevent the adsorption of BSA (protein negatively charged at physiological pH) and lysozyme (protein positively charged at physiological pH). We showed that BSA interaction with SiO2NPs is enthalpically governed. On the other hand, functionalization of silica nanoparticles with PEG and SBS completely prevented BSA adsorption. However, these functionalized nanoparticles presented a negative zeta potential and were not able to suppress lysozyme anchoring due to strong nanoparticle-protein electrostatic attraction. Due to the similarity of BSA with Human Serum Albumin, this investigation bears a resemblance to processes involved in the phenomenon of protein corona in human blood, producing information that is relevant for the future biomedical use of functionalized nanoparticles.

Comparative study on the interaction of oxyresveratrol and piceatannol with trypsin and lysozyme: binding ability, activity and stability.

Natural polyphenols showing a variety of beneficial effects will interact with multiple proteases after administration. The interactions of oxyresveratrol and piceatannol with trypsin and lysozyme were investigated using fluorescence spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, differential scanning calorimetry and molecular docking. Fluorescence quenching results and UV-vis absorption difference spectra revealed that the quenching process was a static mode initiated by ground-state complex formation. The different binding ability of oxyresveratrol and piceatannol with trypsin and lysozyme was discussed based on their different molecular structures. Moreover, the major driving force for the binding process was elucidated as hydrogen bonding and van der Waals forces by the negative enthalpy and entropy changes. Synchronous fluorescence, three-dimensional fluorescence and circular dichroism spectral analysis suggested that the binding of oxyresveratrol and piceatannol to trypsin and lysozyme induced some microenvironmental and conformational changes of the two enzymes. The thermal stability of the enzymes in the presence of polyphenols was studied based on the change in melting temperature by differential scanning calorimetry. The above experimental results were validated by the protein-ligand docking studies which showed the location of the two ligands in the enzymes and the surrounding amino acid residues. Furthermore, enzyme activity assays indicated that the enzymatic activity of trypsin and lysozyme was inhibited by oxyresveratrol and piceatannol. The effect of trypsin and lysozyme on the antioxidant activity and stability of oxyresveratrol and piceatannol was also investigated. In conclusion, the comparative study on the interaction of oxyresveratrol and piceatannol with trypsin and lysozyme showed that the positions of hydroxyl groups of the polyphenols had an important influence on their interaction with enzymes and their antioxidant activity and stability as well as the enzyme activities. The obtained results are expected to provide a theoretical basis for the application of polyphenols in functional foods and pharmaceuticals.

Lysozyme-Assisted Photothermal Eradication of Methicillin-Resistant Staphylococcus aureus Infection and Accelerated Tissue Repair with Natural Melanosome Nanostructures.

Patients often face the challenge of antibiotic-resistant bacterial infections and lengthy tissue reconstruction after surgery. Herein, human hair-melanosome derivatives (HHMs), comprising keratins and melanins, are developed using a simple "low-temperature alkali heat" method for potentially personalized therapy. The mulberry-shaped HHMs have an average width of ∼270 nm and an average length of ∼700 nm, and the negatively charged HHMs can absorb positively charged Lysozyme (Lyso) to form the HHMs-Lyso composites through electrostatic interaction. These naturally derived biodegradable nanostructures act as exogenous killers to eliminate methicillin-resistant Staphylococcus aureus (MRSA) infection with a high antibacterial efficacy (97.19 ± 2.39%) by synergistic action of photothermy and "Lyso-assisted anti-infection" in vivo. Additionally, HHMs also serve as endogenous regulators of collagen alpha chain proteins through the "protein digestion and absorption" signaling pathway to promote tissue reconstruction, which was confirmed by quantitative proteomic analysis in vivo. Notably, the 13 upregulated collagen alpha chain proteins in the extracellular matrix (ECM) after HHMs treatment demonstrated that keratin from HHMs in collagen-dependent regulatory processes serves as a notable contributor to augmented wound closure. The current paradigm of natural material-tissue interaction regulates the cell-ECM interaction by targeting cell signaling pathways to accelerate tissue repair. This work may provide insight into the protein-level pathways and the potential mechanisms involved in tissue repair.

Physicochemical mechanisms of different biopolymers' (lysozyme, gum arabic, whey protein, chitosan) adsorption on green tea extract loaded liposomes.

Having various domains of applicability, liposomes have been the issue of many studies since 1960s. Kinetically stable nature of liposomes required incorporation of other substituents to gain storage stability and interaction of liposomes with polymers, electrolytes, proteins or lipids still requires further investigation to explain the underlying mechanism. In this study, polyphenol-rich green tea extract was encapsulated into liposomes by means of microfluidization in two different aqueous media (pH = 3.8 acetate buffer and pH = 6.5 distilled water). Antioxidant loaded vesicles were further mixed with anionic biopolymers (gum arabic, whey protein) and cationic biopolymers (lysozyme, chitosan) separately. The physical and chemical interactions between liposomes and biopolymers were rationalized by particle size, zeta potential, transmission electron microscopy, total phenolic content and antioxidant activity measurements during 28-days storage at 4 °C. Experimental results indicated that the biopolymer incorporated liposomes showed better stability compared to control liposomes during storage, developing resistance against changes in particle size and zeta potential. On the other hand, biopolymer interaction mechanisms were shown to be different for different biopolymers. As was also proved by transmission electron microscopy, lysozyme was absorbed into the liposomes while gum arabic, whey protein and chitosan were adsorbed on the vesicle surface to shield green tea extract loaded liposomes.

Equilibrium Protein Adsorption on Nanometric Vegetable Oil Hybrid Film/Water Interface Using Neutron Reflectometry.

Nanofilms of about 2 nm thickness have been formed at the air-water interface using functionalized castor oil (ICO) with cross-linkable silylated groups. These hybrid films represent excellent candidates for replacing conventional polymeric materials in biomedical applications, but they need to be optimized in terms of biocompatibility, which is highly related to protein adsorption. Neutron reflectivity has been used to study the adsorption of two model proteins, bovine serum albumin and lysozyme, at the silylated oil (ICO)-water interface in the absence and presence of salt at physiologic ionic strength and pH and at different protein concentrations. These measurements are compared to adsorption at the air-water interface. While salt enhances adsorption by a similar degree at the air-water and oil-water interfaces, the impact of the oil film is significant with adsorption at the oil-water interface 3-4-fold higher compared to that at the air-water interface. Under these conditions, the concentration profiles of the adsorbed layers for both proteins indicate multilayer adsorption. The thickness of the outer layer (oil side) is close to the dimension of the minor axis of the protein molecule, ∼30 Å, suggesting a sideway orientation with the long axis parallel to the interface. The inner layer extends to 55-60 Å. Interestingly, in all cases, the composition of the oil film remains intact without significant protein penetration into the film. The optimal adsorption on these nanofilms, 1.7-2.0 mg·m-2, is comparable to the results obtained recently on thick solid cross-linked films using a quartz crystal microbalance and atomic force microscopy, showing in particular that adsorption at these ICO film interfaces under standard physiological conditions is nonspecific. These results furnish useful information toward the elaboration of vegetable oil-based nanofilms in direct nanoscale applications or as precursor films in the fabrication of thicker macroscopic films for biomedical applications.

Synthesis of magnetic chitin-adsorbent for specific proteins.

A simple, convenient and inexpensive method for the preparation of magnetic chitin composite, in which magnetite particles are densely covered with the polysaccharide shell has been developed. Two-step procedure for magnetic chitin preparation includes: (i) adsorption of chitosan onto magnetite particles and (ii) N-selective acetylation of chitosan to produce magnetic chitin. The composite combines the magnetic properties of magnetite and the adsorption properties of chitin. The synthesized magnetic chitin is an efficient adsorbent of ß-d-GlcNAc-specific lectins and lysozyme. The adsorption capacity of magnetic chitin for wheat germ (Triticum vulgaris) and potato (Solanum tuberosum) lectins was in the range of 67-86 mg g-1 of the adsorbent. Magnetic chitin showed the high capacity for enzyme-lysozyme. Synthesized adsorbent is environmentally friendly and recyclable. Magnetic chitin may be used as a promising multi-purpose adsorbent of pollutants of organic or inorganic nature.

Supramolecular nanoassembly of lysozyme and α-lactalbumin (apo α-LA) exhibits selective cytotoxicity and enhanced bioavailability of curcumin to cancer cells.

Hybrid supramolecular spherical nanoassembly of hen egg white lysozyme and bovine apo α lactalbumin (SNLYZ-BLA) was prepared with a mean size of ˜55.2 nm using an optimized desolvation method via chemical crosslinking. The nanoassembly, SNLYZ-BLA demonstrated dose-dependent reactive oxygen species (ROS) mediated cytotoxicity in multiple cancer cells such as MCF-7, MDA-MB231, HeLa and MG 63. It also demonstrated high loading capacity of a phytochemical based anticancer agent, curcumin (248.8 mg/g) and target-based pH-responsive in vitro drug release with around 85.8% curcumin release observed under acidic condition. Moreover, curcumin loaded SNLYZ-BLA (SNLYZ-BLA-CUR) induced cell viability reduction in all cancer cells including mouse melanoma (B16F10) by more than 90% within 24 h. Further, SNLYZ-BLA and SNLYZ-BLA-CUR when conjugated with folic acid enhanced the cytotoxicity via folate receptor-based targeting. Both drug loading and release induced conformational change and folding reconstitution of the protein nano-assembly, respectively, which made the whole system an efficient therapeutic agent that works via a dual mode of action. We demonstrated that SNLYZ-BLA and SNLYZ-BLA-CUR were highly biocompatible in vitro. Therefore, our supramolecular protein nanoassembly loaded with curcumin could emerge as a comprehensive cancer therapeutics that acts via a strategic mode of dual therapeutic mechanisms.

Mass Transfer of Proteins in Aqueous Two-Phase Systems.

Aqueous Two-Phase Extraction is known to be a gentle separation technique for biochemical molecules where product partitioning is fast. However, the reason for the high mass transfer rates has not been investigated, yet. Many researchers claim that the low interfacial tension facilitates the formation of very small droplets and with it a large interfacial area causing a fast partitioning. However, an experimental evidence for this hypothesis has not been published yet. In this study, the mass transfer coefficients of two proteins, namely lysozyme and bromelain, were determined by providing a defined interfacial area for partitioning. Compared to low molecular weight solutes the mass transfer coefficient for the proteins investigated was small proving for the first time that the large interfacial area and not fast diffusion seems to be the reason for fast protein partitioning.

Aromatic SOFAST-HMBC for proteins at natural 13C abundance.

We propose here SOFAST-HMBC as a new complementary NMR tool for aromatic side chain assignment of protein samples at natural 13C abundance. The characteristic peak patterns detected in SOFAST-HMBC for each aromatic side chain allow straightforward assignment of all protons and carbons (including quaternary ones) of the aromatic ring, and for tyrosine and phenylalanine, connection to the CB of the aliphatic chain. The performance of SOFAST-HMBC is demonstrated for three small proteins (7-14 kDa) at millimolar sample concentration using modern high-field NMR instruments equipped with cryogenically cooled probes. Despite the low amount of NMR-active 13C nuclei in these samples, 1H-13C multiple-bond correlation spectra of good quality were obtained in reasonable experimental times of typically less than 24 h.

Effect of storage on long-term stability of salivary α-amylase, lysozyme, lactate dehydrogenase, calcium and phosphorus in dogs.

Research or diagnostic conditions may require the storage of salivary samples for long periods before analysis is processed. The aim of this study was to evaluate the stability of canine salivary α-amylase, lysozyme, lactate dehydrogenase (LDH), and total calcium and phosphorus after storage for 1, 3 and 6 months at -20°C. Available saliva samples were 75 immediately after collection (T0) and 46 at 1 month (T1), 31 at 3 months (T3) and 18 at 6 months (T6) of storage, according to the number of aliquots collected from each saliva sample. Compared to T0, LDH declined by 92.3% after 1 month of storage (P<0.001), whereas lysozyme concentration significantly decreased as storage time increased (-29.3%, -43.4% and -59.1% at T1, T3, and T6 respectively, P<0.001). Amylase maintained basically the same concentration for the entire experimental period, whereas total calcium and phosphorus concentration decreased over time (calcium, P<0.001). Assessing the long-term stability of canine salivary analytes stored at -20°C may have important implications in diagnosis and research.