Wines in contact with oak wood: the impact of the variety (Carménère and Cabernet Sauvignon), format (barrels, chips and staves), and aging time on the phenolic composition.

BACKGROUND: This study characterized the flavonoid and nonflavonoid phenolic composition of Carménère and Cabernet Sauvignon wines that were in contact with barrels, chips, and staves during a 12 month aging period. The wines were evaluated by spectrophotometric (for total phenols, anthocyanins and tannins, colorant intensity, hue, CIELab parameters, and fractionation into mono-, oligo-, and polymers of proanthocyanidins) and high-performance liquid chromatography diode array detection analyses (for ellagitannins, gallotannins, anthocyanins, and low molecular weight phenols). RESULTS: Wines in contact with oak wood presented a strong enrichment with nonflavonoid compounds, such as caffeic, gallic, and ellagic acids and ellagitannins. Wines in contact with staves stood out for the increased presence of total phenols, vanillic acid, and higher color intensity, whereas wines aged in contact with chips showed large contents of proanthocyanidin gallates. Wines aged in barrels exhibited high contents of ellagitannins and ethyl gallates. The effect of wood on the phenolic composition was mostly associated with the original and intrinsic characteristics of each grape variety. CONCLUSION: Extraction of phenolic compounds from oak wood during wine aging is closely related to the wood format, grape variety (Carménère or Cabernet Sauvignon), and aging time. The final effect of wood on wine would be related not just to the transference of polyphenols from wood, but also to structural modifications of grape polyphenols. © 2018 Society of Chemical Industry.

Proteomic Analysis of Exosomes and Exosome-Free Conditioned Media From Human Osteosarcoma Cell Lines Reveals Secretion of Proteins Related to Tumor Progression.

Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc.

Progress in tear microdesiccate analysis by combining various transmitted-light microscope techniques.

BACKGROUND: Tear desiccation on a glass surface followed by transmitted-light microscopy has served as diagnostic test for dry eye. Four distinctive morphological domains (zones I, II, III and transition band) have been recently recognized in tear microdesiccates. Physicochemical dissimilarities among those domains hamper comprehensive microscopic examination of tear microdesiccates. Optimal observation conditions of entire tear microdesiccates are now investigated. One-µl aliquots of tear collected from individual healthy eyes were dried at ambient conditions on microscope slides. Tear microdesiccates were examined by combining low-magnification objective lenses with transmitted-light microscopy (brightfield, phase contrasts Ph1,2,3 and darkfield). RESULTS: Fern-like structures (zones II and III) were visible with all illumination methods excepting brightfield. Zone I was the microdesiccate domain displaying the most noticeable illumination-dependent variations, namely transparent band delimited by an outer rim (Ph1, Ph2), homogeneous compactly built structure (brightfield) or invisible domain (darkfield, Ph3). Intermediate positions of the condenser (BF/Ph1, Ph1/Ph2) showed a structured roughly cylindrical zone I. The transition band also varied from invisibility (brightfield) to a well-defined domain comprising interwoven filamentous elements (phase contrasts, darkfield). CONCLUSIONS: Imaging of entire tear microdesiccates by transmitted-light microscopy depends upon illumination. A more comprehensive description of tear microdesiccates can be achieved by combining illumination methods.

Phenolic composition and antioxidant capacity of pomaces from four grape varieties (Vitis vinifera L.).

BACKGROUND: Phenolic compounds are widely distributed secondary metabolites in plants usually conferring them with unique taste, flavour and health-promoting properties. In fruits of Vitis vinifera L., phenolic composition is highly dependent on grape variety. Differential extraction of these compounds from grapes during winemaking is critically associated with wine quality. By-products of winemaking, such as grape pomace, can contain significant amounts of polyphenols. However, information concerning the varietal effect on wine grape pomace is scarce. In this study, pomaces from Sauvignon Blanc (SB), Chardonnay (CH), Cabernet Sauvignon (CS) and Carménère (CA) grape varieties were characterized spectroscopically and by HPLC-DAD analysis. RESULTS: White grape pomaces (SB and CH) presented higher antioxidant capacities and higher contents of total phenols and total proanthocyanidins compared with red grape pomaces (CS and CA), whereas the latter showed much higher anthocyanin levels and colour intensities. Concentrations of monomeric proanthocyanidins and low-molecular-weight phenols in the four grape pomace varieties were significantly different. CONCLUSION: Grape pomaces from four varieties showed high but diverse contents of polyphenols and antioxidant capacities. Thus grape pomaces represent an important potential source of polyphenols, which could be useful for nutritional and/or pharmacological purposes.

Dynamics of tear fluid desiccation on a glass surface: a contribution to tear quality assessment.

BACKGROUND: Fern-like crystalloids form when a microvolume of tear is allowed to dry out at ambient conditions on a glass surface. Presence of crystalloids in tear "microdesiccates" is used to evaluate patients with Dry-Eye disease. This study aims to examine morphologically the desiccation process of normal tear fluid and to identify changes associated with accelerated tear evaporation. Tear microdesiccates from healthy (Non-Dry Eye) and Dry Eye subjects were produced at ambient conditions. Microdesiccate formation was monitored continuously by dark-field video microscopy. Additionally, accelerated desiccation of tear samples from healthy subjects was conducted under controlled experimental conditions. Particular morphological domains of tear microdesiccates and their progressive appearance during desiccation were compared. RESULTS: In normal tear microdesiccates, four distinctive morphological domains (zones I, II, III and transition band) were recognized. Stepwise formation of those domains is now described. Experimentally accelerated desiccation resulted in marked changes in some of those zones, particularly involving either disappearance or size reduction of fern-like crystalloids of zones II and III. Tear microdesiccates from Dry Eye subjects may also display those differences and be the expression of a more synchronous formation of microdesiccate domains. CONCLUSION: Morphological characteristics of tear microdesiccates can provide insights into the relative rate of tear evaporation.

Phenolic composition and mouthfeel characteristics resulting from blending Chilean red wines.

BACKGROUND: The blending of wine is a common practice in winemaking to improve certain characteristics that are appreciated by consumers. The use of some cultivars may contribute phenolic compounds that modify certain characteristics in blended wines, particularly those related to mouthfeel. The aim of this work was to study the effect of Carménère, Merlot and Cabernet Franc on the phenolic composition, proanthocyanidin profile and mouthfeel characteristics of Cabernet Sauvignon blends. RESULTS: Significant differences in chemical composition were observed among the monovarietal wines. Separation using Sep-Pak C18 cartridges revealed differences in the concentration but not in the proportion of various proanthocyanidins. Blending reduced polyphenol concentration differences among the various monovarietal wines. Although no major overall differences were observed after blending the monovarietal wines, this oenological practice produced clear differences in mouthfeel characteristics in such a way that the quality of the perceived astringency was different. CONCLUSION: This study showed that the use of a particular wine variety (Cabernet Sauvignon) in a higher proportion in wine blending produced blends that were less differentiable from the monovarietal wine, owing to a suppression effect, producing an apparent standardization of the wines regarding chemical composition.

Different physicochemical interactions between varietal wines and human saliva: Correspondence with astringency.

Astringency corresponds to the sensation of dryness and roughness that is experienced in the oral cavity in association with the interaction between salivary proteins and food polyphenols. In this study, the phenolic composition of seven varietal wines, the intensity of astringency they evoke and the physicochemical reactivity of these wines with whole human saliva were evaluated. Phenolic composition of wines was characterized by spectrophotometry and HPLC chromatography. Intensity of astringency was evaluated by trained sensory panels. Saliva from a single volunteer subject was used to assess wine-saliva interactions. To this end, binary mixtures were produced at different v/v wine/saliva ratios and each of them assayed for the ability of the salivary protein to diffuse on a cellulose membrane (diffusion test) and to remain in solution (precipitation test). Physicochemical reactivities between wine components and the protein fraction of saliva were contrasted against the astringency and the phenolic profile of each varietal wine. The study supports the view that astringency depends on physicochemical interactions between two complex matrices -wine and saliva- and not between some of their particular components.

Microdesiccates produced from normal human tears display four distinctive morphological components.

Desiccation of human tears on glass surfaces results in fern-like crystalloids. This phenomenon has been associated with tear normality (Tear Ferning Test, TFT) and is used as a diagnostic aid to evaluate patients with Dry-Eye disease. However, TFT is focused on the assessment of only a minor fraction of desiccated tear samples and considers only the relative abundance and density of fern-like crystalloids. The aim of this study was to characterize morphologically entire desiccated micro volumes of tears from healthy donors. Tear samples were collected from 23 healthy young adult volunteers. Tear aliquots (1-3 µL) were allowed to dry on glass surfaces under ambient conditions of temperature (15-25°C) and relative humidity (40-45%). Dry samples were analyzed by dark-field microscopy. Morphometric data were acquired with Image J software. Tear volume was positively correlated with both area and time of desiccation. Morphological features of multiple microdesiccates produced from a single subject displayed striking similarities whereas tear microdesiccates from different healthy subjects displayed consistent differences but shared a common general design. This design may be mostly represented by the occurrence of four distinctive zones, named as zones I, II, III and Transition band. The main features of these zones are described.

Effect of inert gas and prefermentative treatment with polyvinylpolypyrrolidone on the phenolic composition of Chilean Sauvignon blanc wines.

BACKGROUND: Sauvignon blanc wines are produced under a wide variety of winemaking conditions, some of which include different fruit-ripening levels, cold soaks and the use of fining agents and inert gases. Anecdotal evidence suggests that sensory variations among these wines may have to do with their phenolic composition and concentration. Therefore the aim of this work was to study the effects of different winemaking conditions typically used in Chile on the phenolic composition and concentration of Sauvignon blanc wines. RESULTS: The use of an inert gas (CO2) in winemaking produced differences in the proportion of proanthocyanidin fractions. A higher concentration of flavan-3-ol monomers resulted from winemaking in the presence of inert gas. This condition also produced a higher content of total phenols and low-molecular-weight phenolic compounds. Low doses of polyvinylpolypyrrolidone (PVPP) in the prefermentative treatments produced wines with a higher content of phenolic compounds. Under these conditions a higher content of polymeric proanthocyanidins was observed. CONCLUSION: Different winemaking conditions modified the concentration and proportion of proanthocyanidin fractions and the global phenolic composition of the resulting white wines. This should be taken into account by the wineries producing these wines.

Paper chromatography approach for the assessment of interaction between red wine and whole saliva.

In-mouth interaction of red wine compounds with salivary proteins is a primary event allegedly responsible for eliciting the mouth-feel sensation of astringency. Those interactions have been currently associated with precipitation of salivary protein/polyphenol complexes. However, such single physicochemical evidence for interaction does not account for the complexity of astringency. This study aimed to develop a paper chromatography method to assess interactions between red wine and the salivary protein fraction using stepwise series of red wine/saliva binary mixtures from 100% wine to 100% saliva ("Alpha and Omega series"). Aliquots of each one of the mixtures were spotted on a cellulose membrane to scrutinize independently the distribution areas of wine components (naturally pink-colored) and salivary protein (stained blue in Coomassie Brilliant R-250). This double target detection revealed interactions between saliva and red wine components along most of the quantitative Alpha and Omega series, a point of equivalence corresponding to maximum interactivity for both complex reactants and a non-diffusible sub-fraction of saliva displaying the highest interactivity. The results indicate a novel way to assess quantitatively physicochemical interactions between red wines and human saliva but also provide new lights to approach the identification of molecular salivary structures involved in triggering astringency.

Arginine 66 residue of Fur is required for the regulatory function of this protein in the acid adaptation mechanism of Helicobacter pylori.

BACKGROUND: Helicobacter pylori colonizes the gastric mucosa and must survive the acid pH of that environment. Like other enteric bacterial pathogens, including Salmonella enterica, H. pylori develops an acid tolerance response that is dependent on the function of the transcriptional regulator protein Fur. OBJECTIVE: To explore by site-directed mutagenesis whether two particular amino acid residues in the amino acid sequence of the H. pylori Fur protein, arginine 66 and histidine 99, are involved in the acid response mechanism in this bacterium. MATERIALS AND METHODS: Complementation assays in Escherichia coli H1780 (fur null mutant) both with plasmids carrying the H. pylori fur gene bearing substitution mutations R66A or H99A or R66A/H99A and with the H. pylori Fur-R66A mutant were conducted. Wild-type and mutated Fur proteins from H. pylori were assayed by using the fiu::lacZ reporter gene in the E. coli H1780 heterologous system at various pH and iron concentrations. RESULTS: Both bacterial growth and repression of the reporter gene were impaired under acid conditions in E. coli H1780 complemented with pUC19-fur-R66A. Also, in the H. pylori Fur-R66 strain bacterial growth and speA gene expression were impaired under acid conditions. CONCLUSIONS: Arginine 66 but not histidine 99 in H. pylori Fur is required for the regulatory function of the Fur protein in the acid adaptation mechanism of the bacterium.

Dry Eye and Visual Display Terminal-Related Symptoms among University Students during the Coronavirus Disease Pandemic.

PURPOSE: To evaluate dry eye (DE) and subjective visual display terminal (VDT)-related symptoms in university students who moved their classes online due to the COVID-19 pandemic. METHODS: Cross-sectional study of students who were taking online classes. In May 2020, the participants completed a Dry Eye Questionnaire (DEQ-5) and a self-report survey, which included demographics, medical history, information on the use of VDT and presence of VDT-related symptoms. Participants were classified as having mild/moderate (7-12) or severe (>12) DE symptoms based on their DEQ-5 score. The associations between severe DE symptoms and relevant factors were also evaluated. RESULTS: The data of 1450 eligible students were analyzed. The mean age of the participants was 21.1 (2.7) years. 42.8% of the participants had mild/moderate DE symptoms, whereas 34.7% had severe symptoms. Associated factors for severe DE were female sex (OR = 2.57, CI [1.97-3.35]), allergic disease (OR = 1.63, CI [1.24-2.13]), previous dry eye diagnosis (OR = 13.49, CI [7.10-25.63]), keratoconus (OR = 5.56, CI [1.27-24.44], contact lens use (OR = 1.77, CI [1.24-2.53]) and duration of VDT use (OR = 1.02, CI [1.01-1.05]). Prior to the pandemic, the mean reported duration of VDT use was 9.8 (4.7) hours; this increased to 15.9 (5.8) hours during the online classes (p < .001). 80.6% of the participants reported a global increase in VDT-related symptoms. CONCLUSION: Students taking online classes had a high frequency of DE symptoms. They also reported a significant increase in VDT-related symptoms. DE should be considered as an emerging health problem among the young population, which is probably related to the recent changes in lifestyle.

tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori.

About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat) was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR) by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.

Tetracycline resistance in Chilean clinical isolates of Helicobacter pylori.

OBJECTIVES: Since high-level tetracycline resistance in Helicobacter pylori has been associated with a AGA926-928-->TTC substitution in the 16S rRNA genes rrnA/B, the aim of the study was to screen for tetracycline resistance in H. pylori clinical isolates obtained from Santiago, Chile by using a recently reported molecular assay. METHODS: A PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the conserved 535 bp region of the H. pylori 16S rRNA genes rrnA/B (between nucleotides 710 and 1245) using HinfI was followed by DNA sequencing of the same fragment obtained from tetracycline-resistant H. pylori clinical isolates. RESULTS: The PCR-RFLP assay revealed that the tetracycline-resistant H. pylori isolates lacked the AGA926-928-->TTC substitution. In contrast, DNA sequencing of the 535 bp PCR fragment from 11 tetracycline-resistant H. pylori Chilean clinical isolates showed an association of low-level tetracycline resistance with 1 bp (A928C) or 2 bp (AG926-927-->GT and/or A926G/A928C) substitutions in both 16S rRNA genes. CONCLUSIONS: The PCR-RFLP (HinfI) assay alone is unreliable for the detection of tetracycline resistance in Chilean clinical isolates of H. pylori. To that end, it must be complemented by sequencing of the 535 bp PCR fragment.

Chromosomal size conservation through the cell cycle supports karyotype stability in Trypanosoma cruzi.

The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.

A protein dye-binding assay on cellulose membranes for tear protein quantification: use of conventional schirmer strips.

PURPOSE: To develop a method to quantify tear protein concentration with the sensitivity to measure this variable in the restricted volumes of single human tear samples. METHODS: Aliquots of tear fluid from healthy subjects and a solution of standard bovine serum albumin (BSA) were spotted on cellulose membranes. Membranes were fixed, stained for protein with Coomassie blue, and washed until they displayed clear backgrounds. Stained spots were excised and eluted in a defined volume of methanol-ammonia, and the absorbance was determined spectrophotometrically at 610 nm. Membranes were calibrated by calculating their apparent thickness from the areas of stained spots and the corresponding aliquot volumes of either tear fluid or BSA solution. RESULTS: In our dye-binding assay, absorbance (0-1.00 OD) was found to have a linear relation with tear fluid volume (1-7 microL). In a study involving samples from 33 healthy subjects, aliquots (3 microL) of tear fluid were found to yield absorbances in the linear range. Protein concentrations in tear fluid were found to be distributed over the range of 2.20-6.37 mg/mL (mean, 4.11 +/- 1.00 mg/mL) with no apparent sex differences. The assay can be applied successfully to quantify protein concentrations in tear fluid by using calibrated Schirmer strips after a tear test. Electrophoretic profiles of proteins present in tear fluid sampled from different healthy individuals were nearly identical when normalized for protein load by using this method. CONCLUSIONS: The protein dye-binding assay we developed by using cellulose membranes or Schirmer strips is an efficient and convenient method for measuring tear protein concentration.

Zone I of Tear Microdesiccates Is a Lipid-Containing Structure.

PURPOSE: Morphological features of tear microdesiccates on glass surfaces have been associated with tear fluid status. Tear-film lipids play a critical role in the pathophysiology of some ocular surface disorders. Tear microdesiccates display 4 distinctive morphological domains (zones I, II, III, and transition band). In this study, we investigated the lipid location in tear microdesiccates. METHODS: Tear from individual healthy eyes (assessed by symptoms, signs, and slit-lamp examination) was collected using absorbing minisponges. One-µL aliquots were allowed to dry under ambient conditions on microscope slides. Tear microdesiccates were examined by various transmitted light microscopy methods. Tear lipids were located both by partition experiments using 2 lipophilic dyes (Oil red O and Nile blue A) mixed with tear fluid under conditions preserving morphological features of microdesiccates and by assessing the effect of 2 solvents markedly differing in polarity (water and ethanol) on the morphology of particular domains of preformed microdesiccates. RESULTS: During desiccation, both Nile blue A and Oil red O became preferentially located in the outermost domain of tear microdesiccates (zone I) without affecting the formation of major fern-like crystalloids (zones II and III). Low volumes of water drastically affected fern-like crystalloids, whereas the gross morphology of zone I was maintained. Contrarily, ethanol, a less polar solvent, was a fixative for fern-like crystalloids, although it markedly affected the bulk of zone I by extracting liquid droplets out of microdesiccates and visibilizing some filamentous subcomponents. CONCLUSIONS: Zone I is a hydrophobic domain, whereas zones II and III are highly hydrophilic domains of tear microdesiccates. Zone I represents a lipid-rich structure.

Great diversity among commercial inactive dry-yeast based products.

Commercial inactive dry-yeast based (IDYB) products have been shown to impact positively in different ways on the winemaking process, including sensory enhancement. Despite their relevance little information about physicochemical characteristics of individual IDYB products is available. This study aimed to physicochemically characterize a group of ten commercial IDYB products. Organic, protein and carbohydrate contents by spectrophotometric methods, protein diffusion on cellulose membranes and electrophoretic protein profiles were assessed. Interaction of a IDYB product (CP10) with either salivary protein or a proanthocyanidin-rich extract (binary mixtures) or with both of them (ternary mixtures) was also assessed. Marked physicochemical differences were observed among all ten products. CP10 was found to interact with seed extract and salivary protein. Also, as part of CP10-SE complexes, CP10 interacted with the salivary protein to form ternary complexes. Due to their huge diversity, physicochemical characterization of IDYB products before use in winemaking is recommended.

Algorithm approach for revision surgery following late-onset bleb complications after trabeculectomy: long-term follow-up.

PURPOSE:: The aim of this study was to introduce a reproducible algorithm for the surgical management of late-onset (>2 months) bleb complications after trabeculectomy with mitomycin C. METHODS:: We performed a retrospective review of eyes treated using a reproducible algorithm approach by a single surgeon for the surgical management of late-onset bleb complications from July 2006 to April 2014. Exclusion criteria were bleb revision with less than 3 months of follow-up or bleb revision combined with other glaucoma procedures at the time of surgery. Success was evaluated using the Kaplan-Meier survival method and defined as achieving all of the following criteria: primary surgery indication resolved, no additional surgery required for decreasing the intraocular pressure (IOP), and IOP of ≥6 mmHg and ≤18 mmHg. RESULTS:: Twenty-three eyes from 20 patients were evaluated. Indications for bleb revision were hypotonic maculopathy (47.8%), bleb leak (30.4%), and dysesthetic bleb (21.7%). The overall primary outcome success rate calculated using the Kaplan-Meier survival method was 65.2% at 48 months. When the IOP target was changed to ≤15 mmHg, the bleb survival rate was 47.8% at 48 months. At the most recent postoperative visit, 95.7% of eyes had an IOP of ≤15 mmHg and 56.5% were being treated with an average of one medication per eye. One eye (4.3%) required a second bleb revision for persistent hypotony and two eyes required glaucoma surgery to reduce IOP during follow-up. CONCLUSIONS:: An algorithm approach for the surgical management of late-onset bleb complications with a success rate similar to those reported in specialized literature is proposed. Randomized trials are needed to confirm the best surgical approach.

Use of polyurethane minisponges to collect human tear fluid.

PURPOSE: To characterize a method of tear collection based on the use of amphiphilic polyurethane absorbing minisponges. METHODS: Tear fluid was collected from 17 healthy volunteers. A preweighed polyurethane dry minisponge was laid on the margin of the lower eyelid. Once wet (5-10 minutes), the fluid was transferred to a preweighed Eppendorf tube after squeezing the sponge by centrifugation. The amount of fluid absorbed and fluid recovered were determined by reweighing the sponge and the tube after absorption and centrifugation steps, respectively. The fluid was qualitatively characterized by electrophoretic polypeptide profiling in Coomassie blue-stained SDS-polyacrylamide gels. RESULTS: Per eye, 14.6 +/- 5.3 microL tear fluid was collected. That volume was about 90% of the fluid absorbed by polyurethane minisponges, almost doubling the fraction recovered from other more hydrophilic absorbing polymers. Major bands characterizing the electrophoretic profile of this fluid were those of 79, 66, 27, 18, and 14 kd. This profile was indistinguishable from that of tear fluid aspirated into glass microcapillaries. Tear fluid collected simultaneously from both eyes displayed the same profiles. Successive tear samples from a single eye showed the same profile except for the 66-kd band, which increased steadily as collection proceeded. Tear donors rarely complained of discomfort. CONCLUSIONS: Tear collection by absorbing polyurethane minisponges is highly advantageous in efficiency (recovery) and reproducibility (invariant electrophoretic polypeptide profiles). Tear donor comfort, simultaneous bilateral collection, and collections from several donors at once are additional major advantages of this collection method in studies involving single subjects and populations in health and disease.