Mechanisms of superficial micropunctate corneal staining with sodium fluorescein: The contribution of pooling

Purpose

To establish if sodium fluorescein (SFL) dye accumulation within intercellular spaces on the ocular surface contributes to the appearance of superficial punctate corneal staining.

Methods

Thirteen subjects bilaterally wore PureVision™ lenses that had been pre-soaked in ReNu MultiPlus^® multipurpose solution. After 1 h of lens wear, corneal staining with SFL was assessed using a standard slit-lamp technique. Participants who presented with bilateral, corneal staining were selected for further evaluation. A randomly selected eye was rinsed with saline three times. Fellow eyes (control) received no rinsing. After each rinse, the appearance of SFL staining was recorded without any further instillation of the dye. To eliminate any confounding effects of staining due to residual fluorescein in the tear menisci, corneal staining was induced in freshly excised, isolated, rabbit eyes by topical administration of 0.001% PHMB and staining, rinsing and grading were performed as above.

Results

Nine out of 13 subjects presented with bilateral diffuse corneal staining (mean grade ± SD: 2.4 ± 0.7). The mean staining grades in test and control eyes respectively after each of the three rinses were (1) 2.41 ± 0.41, 2.25 ± 0.69 (p = 0.9); (2) 2.34 ± 0.79, 2.1 ± 0.83 (p = 0.8); and (3) 1.71 ± 0.65, 1.60 ± 0.79 (p = 0.6) there was no significant reduction in staining with rinsing (p > 0.05) and no difference was observed between test and control eyes at any sampling-point. Similar observations made in ex vivo rabbit eyes replicated these results.

Conclusions

Pooling or accumulation of SFL solution within intercellular spaces does not appear to contribute to the appearance of superficial micropunctate corneal staining.     

Prebiotic effect of resistant starch from Vigna unguiculata (L.) Walp. (cowpea) using an in vitro simulated digestion model

There has been growing interest in the use of resistant starch (RS) as a prebiotic for physiological benefits and to reduce the risk of chronic diseases. In this study, RS was isolated from samples of five cowpea cultivars (DT129-4, TVU 11424, PAN 311, Fahari and Bechuana White) seeds to determine their potential prebiotic effects, and proximate and mineral composition. Results obtained showed samples of five cowpea cultivars to be high in protein (18.0–28.7%) and carbohydrates (54.9–66.0%) and low in fat (0.6–3.7%). The RS from cowpea samples ranged between 9.3 and 12.1% with cultivar DT129-4 having the highest amount. Fermentation of RS using mixed and pure cultures was done, and bacterial growth was monitored over a 24-h period. Resistant starch isolated from all cowpea samples fulfilled the criteria to be classified as a prebiotic which have a wide range of beneficial effects on the host, with cultivar DT129-4 showing the most prebiotic potential amongst cowpea samples tested.     

Analysis of Escherichia coli cell damage induced by HPCD using microscopies and fluorescent staining

Cellular damage of Escherichia coli (E. coli) induced by high pressure carbon dioxide (HPCD) at 37-57 °C and 10-30 MPa for 5-75 min was investigated using scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), confocal laser scanning microscopy (CLSM), and fluorospectrophotometer (FSM). HPCD-induced alterations in the morphology and the intracellular organization of E. coli cells was more susceptible to HPCD. A vast majority of HPCD-treated E. coli cells with seemingly intact morphology sustained severe damage in their intracellular organization. CLSM suggested that initial disruption of the outer membrane and later permeabilization of the cytoplasmic membrane of HPCD-treated E. coli cells was a consecutive and progressive process. These results were confirmed by FSM with the probes PI and SYTO 9. The membrane fluidity of HPCD-treated E. coli cells decreased as suggested by increased fluorescence polarization using FSM with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of 37, 42 and 47 °C alone showed no impact on the outer membrane and membrane fluidity of E. coli cells whereas 57 °C alone had greater impact on them. Combined with HPCD, the temperatures of 37, 42 and 47 °C disrupted the outer membrane of E. coli cells without damage to the cytoplasmic membrane and of 57 °C damaged the cytoplasmic membrane, but all these temperatures decreased the membrane fluidity of E. coli cells. Higher temperature increased HPCD-induced outer membrane disruption and the cytoplasmic membrane damage and decreased the membrane fluidity.     

Gastric digestion of milk protein ingredients: Study using an in vitro dynamic model

The coagulation behavior and the kinetics of protein hydrolysis of skim milk powder, milk protein concentrate (MPC), calcium-depleted MPC, sodium caseinate, whey protein isolate (WPI), and heated (90°C, 20 min) WPI under gastric conditions were examined using an advanced dynamic digestion model (i.e., a human gastric simulator). During gastric digestion, these protein ingredients exhibited various pH profiles as a function of the digestion time. Skim milk powder and MPC, which contained casein micelles, formed cohesive, ball-like curds with a dense structure after 10 min of digestion; these curds did not disintegrate over 220 min of digestion. Partly calcium-depleted MPC and sodium caseinate, which lacked an intact casein micellar structure, formed curds at approximately 40 min, and a loose, fragmented curd structure was observed after 220 min of digestion. In contrast, no curds were formed in either WPI or heated WPI after 220 min of digestion. In addition, the hydrolysis rates and the compositions of the digesta released from the human gastric simulator were different for the various protein ingredients, as detected by sodium dodecyl sulfate-PAGE. Skim milk powder and MPC exhibited slower hydrolysis rates than calcium-depleted MPC and sodium caseinate. The most rapid hydrolysis occurred in the WPI (with and without heating). This was attributed to the formation of different structured curds under gastric conditions. The results offer novel insights about the coagulation kinetics of proteins from different milk protein ingredients, highlighting the critical role of the food matrix in affecting the course of protein digestion.     

Carotenoid bioaccessibility from nine raw carotenoid-storing fruits and vegetables using an in vitro model

BACKGROUND: Many techniques exist for processing fruits and vegetables. The impact of these processes on nutritional qualities of the food can be considerable, however. Given the benefits of eating raw foods, nutrient sources need to be identified that deliver substantial benefit without cooking. In this study a survey of carotenoid bioaccessibility was carried out in order to additionally evaluate the impact of their distinctive storage structures (chromoplasts) on bioaccessibility. RESULTS: Per cent carotenoid bioaccessibility varied among the nine raw, whole fruits and vegetables evaluated, with values of 1–39% for lycopene, 18–20% for α‐carotene, 7–49% for β‐carotene, 9–59% for lutein, 4–22% for violaxanthin and 47–96% for phytoene. Per 100 g of food, grapefruit and watermelon imparted the most lycopene (69 and 64 µg respectively), carrot the most α‐carotene (559 µg), β‐carotene (1078 µg), lutein (91 µg) and phytoene (23 mg) and mango the most violaxanthin (177 µg). Digestive stability averaged over 80%, except for the xanthophylls, which exhibited a wider and lower range of stabilities. CONCLUSION: These data identify raw food sources for carotenoid bioaccessibilities comparable to those of other foods accomplished by substantial processing. The information presented here also has application in identifying appropriate plant‐breeding goals and optimal sources for commercial carotenoid isolations. Copyright © 2012 Society of Chemical Industry     

宁夏枸杞体外抗氧化机理研究

目的：研究宁夏枸杞中主要的4种抗氧化物质（枸杞多糖、黄酮、类胡萝卜素和VC）的抗氧化能力,分析它们对枸杞果实总抗氧化能力的贡献率,探索宁夏枸杞果实的体外抗氧化机理。方法：采用FRAP法、DPPH法、ABTS法和清除羟自由基法4种体外抗氧化方法来评价枸杞鲜果的总抗氧化能力。结果：4种方法测得枸杞黄酮对枸杞果实总抗氧化能力的贡献率均大于96%,VC对枸杞果实总抗氧化能力的贡献率均小于4%,高质量浓度的枸杞多糖（642.7170 mg/L）和类胡萝卜素（109.9280 mg/L）检测不到抗氧化能力。结论：枸杞黄酮的抗氧化能力决定着枸杞果实的体外抗氧化能力。     

Survival of probiotic lactobacilli in the upper gastrointestinal tract using an in vitro gastric model of digestion

The aim of this study was to investigate survival of three commercial probiotic strains (Lactobacillus casei subsp. shirota, L. casei subsp. immunitas, Lactobacillus acidophilus subsp. johnsonii) in the human upper gastrointestinal (GI) tract using a dynamic gastric model (DGM) of digestion followed by incubation under duodenal conditions. Water and milk were used as food matrices and survival was evaluated in both logarithmic and stationary phase. The % of recovery in logarithmic phase ranged from 1.0% to 43.8% in water for all tested strains, and from 80.5% to 197% in milk. Higher survival was observed in stationary phase for all strains. L. acidophilus subsp. johnsonii showed the highest survival rate in both water (93.9%) and milk (202.4%). Lactic acid production was higher in stationary phase, L. casei subsp. shirota producing the highest concentration (98.2 mM) after in vitro gastric plus duodenal digestion.     

Evaluation of gastrointestinal solubilization of petroleum hydrocarbon residues in soil using an in vitro physiologically based model

Petroleum hydrocarbon residues in weathered soils may pose risks to humans through the ingestion pathway. To understand the factors controlling their gastrointestinal (GI) absorption, a newly developed experimental extraction protocol was used to model the GI solubility of total petroleum hydrocarbon (TPH) residues in highly weathered soils from different sites. The GI solubility of TPH residues was significantly higher for soil contaminated with diesel than with crude oil. Compared to the solubility of TPH residues during fasted state,the solubility of TPH residues during fat digestion was much greater. Diesel solubility increased from an average of 8% during the "gallbladder empty" phase of fasting (and less than 0.2% during the otherfasting phase) to an average of 16% during fat digestion. For crude oil, the solubility increased from an average of 1.2% during the gallbladder empty phase of fasting (and undetectable during the other fasting phase) to an average of 4.5% during fat digestion. Increasing the concentration of bile salts also increased GI solubility. GI solubility was reduced by soil organic carbon but enhanced by the TPH content.     

An investigation of strong sodium retention mechanisms in nanopore environments using nuclear magnetic resonance spectroscopy

Recent experimental research into the adsorption of various cations on zeolite minerals has shown that nanopore channels of approximately 0.5 nm or less can create an effect whereby the adsorption of ions, especially those that are weakly hydrated, can be significantly enhanced. This enhanced adsorption occurs due to the removal of hydrating water molecules which in turn is caused by the nanopore channel's small size. A new adsorption model, called the nanopore inner-sphere enhancement (NISE) effect, has been proposed that explains this unusual adsorption mechanism. To further validate this model a series of nuclear magnetic resonance (NMR) spectroscopy studies is presented here. NMR spectra were gathered for Na adsorbed on three zeolite minerals of similar chemical composition but differing nanoporosities: zeolite Y with a limiting dimension of 0.76 nm, ZSM-5 with a limiting dimension of 0.51 nm, and mordenite with a limiting dimension of 0.26 nm. The NMR experiments validated the predictions of the NISE model whereby Na adsorbed via outer-sphere on zeolite Y, inner-sphere on ZSM-5, and a combination of both mechanisms on mordenite. The strong Na adsorption observed in these nanoporous minerals conflicts with sodium's general designation as a weak electrolyte.     

Analgesic efficacy of sodium salicylate in an amphotericin B-induced bovine synovitis-arthritis model

This study examined the efficacy of sodium salicylate for providing analgesia in an amphotericin B-induced bovine synovitis-arthritis model using 10 male Holstein calves, 4 to 6 mo old and weighing approximately 250 kg. The study used a repeated measures partial crossover design with 2 phases, consisting of 3 treatment periods within each phase. Calves were blocked by body weight and randomly assigned to the sodium salicylate (50 mg/kg i.v.) or placebo group for phase 1. In period 1, lameness induction was simulated with a needle prick of the coronary band, followed by drug or placebo administration. At predetermined time points, serial blood samples for cortisol and salicylate concentrations, electrodermal activity measurements, heart rates, and pressure mat data were collected. Visual lameness scores were recorded by an observer blinded to treatments. In period 2, lameness was induced with injection of amphotericin B into the distal interphalangeal joint, followed by drug or placebo administration, with sample collection as described previously. In period 3, the drug or placebo was administered to the respective calves with sample collection. After a 10-d washout period, phase 2 was conducted with treatments crossed over between groups. Cortisol and salicylate samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay, respectively. The pharmacokinetic data were analyzed using compartmental analysis. Mean intravenous salicylate apparent volume of distribution was 0.2 ± 0.005 L/kg, total body clearance was 4.3 ± 0.2 mL/min·kg, and elimination half-life was 36.9 ± 1.2 min. The repeated measures data were analyzed based on a univariate split-plot approach with a random effects-mixed model. Differences in stance phase duration and serum cortisol concentration values were seen both between periods and between treatment group × periods; differences in heart rate, contact surface area, and contact pressure values were seen between periods, suggesting that our lameness model was effective. No differences were seen between treatment groups. When analyzed by visual lameness score, differences were seen in heart rate, contact surface area, contact pressure, and cortisol concentrations. Area under the time-effect curves, determined by using the trapezoidal rule, had results similar to the repeated measures data, except for a difference in period for electrodermal activity. This amphotericin B-induced synovitis-arthritis model is a useful tool for studying changes associated with lameness in cattle. Sodium salicylate was not effective in providing analgesia after lameness.     

Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model

The behaviour of cationic lactoferrin-stabilized and anionic β-lactoglobulin (β-lg)-stabilized oil-in-water emulsions (20.0% (w/w) soy oil, 1.0% (w/w) protein) in the presence of simulated intestinal fluid (SIF) containing physiological concentrations of pancreatin (0.0–10.0 mg mL^?1) and/or bile salts (0.0–25.0 mg mL^?1) at 37 °C, pH 7.5 and inorganic salts (39 mm K₂HPO₄, 150 mm NaCl and 30 mm CaCl₂) was investigated. Both emulsions showed a significant degree of coalescence and fatty acid release on mixing with SIF. Appreciably negative ζ-potential values (≥?50 mV) for both types of emulsion droplet at the highest pancreatin/bile salts concentration could be attributed to displacement of and/or binding to the interfacial proteins by bile salts, together with interfacial proteolysis by pancreatin, which enhanced the potential for lipase to act on the hydrophobic lipid core, thus generating free fatty acids and possibly mono- and/or diglycerides at the droplet surface.     

Determination of sodium in processed foodstuffs using a bis(12-crown-4) derivative-based poly(vinyl chloride) membrane electrode

A poly(vinyl chloride) matrix membrane sodium selective electrode based on bis[(12-crown-4)ylmethyl] 2-dodecyl-2-methylmalonate ((12-crown-4)yl = 1,4,7,10-tetraoxacyclododecyl) was utilised for the determination of sodium in various foodstuffs such as soup stocks, instant noodle stocks and milk powders. Good agreement was obtained between the proposed electrode and flame photometric methods. Also, the electrode exhibits minimum interference from potassium, calcium, magnesium, iron(III), copper(II) and glucose, and from the synthetic colours tested.     

Cosmetic efficacy claims in vitro using a three-dimensional human skin model

A tissue engineered human skin equivalent is successfully used for the testing of raw materials and cosmetic formulations. This reconstructed skin is supported by a collagen-glycosaminoglycan-chitosan biopolymer in which human keratinocytes and dermal fibroblasts were co-cultured to form a tissue that closely reproduces the in vivo architecture of normal human skin and takes into account the complex interactions between epidermis and dermis. On the other hand, dermal and epidermal responses can be assessed separately in the dermal or skin equivalent. The three-dimensional model has important advantages compared to monolayer cell cultures and epidermis models in efficacy testing: (i) the possibility of long-term cultivation with repeated application of cream formulations containing bioactives and (ii) the similarity to human skin concerning the interaction between dermis and epidermis. These similarities include the expression of keratinocyte differentiation markers such as cytokeratin 10, filaggrin and transglutaminase, as well as proteins of the basal lamina (laminin, collagen type IV) and extracellular matrix proteins such as elastin. The efficacy of selected bioactives was determined using different endpoints, for example, stimulation of collagen synthesis in the dermal and skin equivalents was shown in comparison to vitamin C as a positive control. On skin equivalents using immunofluorescence techniques we also demonstrated stimulation of the differentiation marker filaggrin, which is important for skin moisturization. The results could be used for claim substantiation, e.g. for the treatment of dry and aged skin.     

Peroxidase (POD) and polyphenol oxidase (PPO) photo-inactivation in a coconut water model solution using ultraviolet (UV)

The interest in coconut water as a beverage is increasing due, not only to its sensory properties, but also to its nutritional characteristics. Even so, several challenges limit its processing, the inactivation of the polyphenol oxidase (PPO) and peroxidase (POD) enzymes being the most important. Although the inactivation of these enzymes has been extensively studied in coconut water, both by conventional and emerging technologies, the technologies evaluated so far are either not effective in the inactivation of these enzymes and/or result in undesirable changes. This work evaluated the photo-inactivation of POD and PPO in a coconut water model solution using ultraviolet radiation (UV). Both enzymes showed continuous inactivation behaviour in relation to the processing time, this being described by a two-portion inactivation kinetics. A possible mechanism for the observed photo-inactivation was proposed, involving steps of molecular unfolding and aggregation. The POD activity after 15 min of processing was ~ 5% of its original value, and reduced to ~ 1% after 30 min of UV processing. After 15 min of processing, PPO activity was ~ 8% of its original value, falling to ~ 2% after 30 min of UV processing. The results obtained highlight the potential use of the ultraviolet radiation to inactivate both enzymes in coconut water.