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Liposomes were used as biomimetic models in capillary electrokinetic chromatography (EKC) for the determination of distribution constants (K D ) of certain local anesthetics and a commonly used preservative. Synthetic liposomes comprised phosphatidylcholine and phosphatidylglycerol phospholipids with and without cholesterol. In addition, ghost liposomes made from red blood cell (RBC) lipid extracts were used as pseudostationary phase to acquire information on how the liposome composition affects the interactions between anesthetics and liposomes. These results were compared with theoretical distribution coefficients at pH 7.4. In addition to 25°C, the distribution constants were determined at 37 and 42°C to simulate physiological conditions. Moreover, the usability of five electroosmotic flow markers in liposome (LEKC) and micellar EKC (MEKC) was studied. LEKC was proven to be a convenient and fast technique for obtaining data about the distribution constants of local anesthetics between liposome and aqueous phase. RBC liposomes can be utilized for more representative model of cellular membranes, and the results indicate that the distribution constants of the anesthetics are greatly dependent on the used liposome composition and the amount of cholesterol, while the effect of temperature on the distribution constants is less significant., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: To analyze the outcome of penetrating keratoplasty (PK) to the first eye for corneal amyloidosis in familial amyloidosis, Finnish type (FAF)., Design: Single-center, retrospective, nonrandomized, interventional, noncomparative case series., Participants: Thirty-one eyes of 31 patients with FAF., Intervention: All patients with FAF who had their first PK in Helsinki University Eye Hospital between January 1, 1990, and August 1, 2011, were identified and a retrospective analysis of the patient charts was performed., Main Outcome Measures: Best spectacle-corrected visual acuity (BCVA), intraoperative and postoperative complications, graft survival, reason for graft failure, and frequency of regrafting., Results: The median follow-up period was 32 months (range, 5-114). After 24 months, the median BCVA was 1.15 on a logarithm of the minimum angle of resolution scale (20/280; mean, 1.1; SD, 0.5) in comparison with the preoperative median BCVA of 1.3 (20/400; mean, 1.3; SD, 0.4). At 24 months, 3 of 18 eyes (17%) had a visual acuity of ≥0.5 (20/63) and 13 of 18 grafts (72%) were clear. Rejection occurred in 6 of 31 primary grafts (19%). Graft failure occurred in 16 of 31 eyes and resulted from surface complications in 11 eyes and additionally from rejection in 5 eyes. Seven eyes needed regrafting (twice in 1 eye). Complications were frequent in the early and late postoperative periods. Presence of preoperative corneal or graft neovascularization was an indicator of a high risk of graft failure and poor visual outcome., Conclusions: In a minority of FAF patients, PK improves vision. Owing to the high failure risk and guarded visual prognosis after PK, it is important that both the surgeon and the patient have realistic expectations. It may be reasonable to limit PK to cases with bilateral advanced disease. It seems reasonable to optimize ocular surface health and to delay PK., (Copyright © 2015 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Hyperosmolarity (HO) imposes a remarkable stress on membranes, especially in tissues in direct contact with the external environment. Our efforts were focused on revealing stress-induced lipid changes that precede the inflammatory cytokine response in human corneal epithelial cells exposed to increasing osmolarity. We used a lipidomic analysis that detected significant and systematic changes in the lipid profile, highly correlated with sodium concentrations in the medium. Ceramides and triglycerides (TGs) were the most-responsive lipid classes, with gradual increases of up to 2- and 3-fold, respectively, when compared with control. The source of ceramide proved to be sphingomyelin hydrolysis, and neutral sphingomyelinase 2 (NSM2) activity showed a 2-fold increase 1 h after HO stress, whereas transcription increased 3-fold. Both TG accumulation and IL-8 secretion were shown to be dependent on ceramide production by specific knock-down of NSM2. In HCE cells, diglyceride acyltransferase 1 was responsible for the TG synthesis, but the enzyme activity had no effect on cytokine secretion. Hence, NSM2 plays a key role in the cellular response to hyperosmolar stress, and its activity regulates both cytokine secretion and lipid droplet formation.

Intravenous lipid emulsion is recommended as treatment for local anesthetic intoxication based on the hypothesis that the lipophilic drug is entrapped by the lipid phase created in plasma. We compared a 15.6 mM 80/20 mol% phosphatidyl choline (PC)/phosphatidyl glycerol (PG)-based liposome dispersion with the commercially available Intralipid® emulsion in a pig model of local anesthetic intoxication. Bupivacaine-lipid interactions were studied by electrokinetic capillary chromatography. Multilamellar vesicles were used in the first in vivo experiment series. This series was interrupted when the liposome dispersion was discovered to cause cardiovascular collapse. The toxicity was decreased by an optimized sonication of the 50% diluted liposome dispersion (7.8 mM). Twenty anesthetized pigs were then infused with either sonicated PC/PG liposome dispersion or Intralipid®, following infusion of a toxic dose of bupivacaine which decreased the mean arterial pressure by 50% from baseline. Bupivacaine concentrations were quantified in blood samples using liquid chromatography/mass spectrometry. No significant difference in the context-sensitive plasma half-life of bupivacaine was detected (p=0.932). After 30 min of lipid infusion, the bupivacaine concentration was 8.2±1.5 mg/L in the PC/PG group and 7.8±1.8 mg/L in the Intralipid® group, with no difference between groups (p=0.591). No difference in hemodynamic recovery was detected between groups (p > 0.05)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In the tear fluid the outermost part facing the tear-air interface is composed of lipids preventing evaporation of the tears. Phospholipid transfer protein (PLTP) mediates phospholipid transfer processes between serum lipoproteins and is also a normal component of human tears. To study whether PLTP plays any functional role in tear fluid we investigated PLTP-deficient mice, applying functional and morphologic analyses under normal housing and experimentally induced dry eye conditions. Aqueous tear fluid production, corneal epithelial morphology, barrier function, and occludin expression were assessed. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression were shown. These pathologic signs were worsened by experimentally induced dry eye both in wild-type and PLTP knock-out mice. Deficiency in the production of tear PLTP in mice is accompanied by corneal epithelial damage, a feature that is typical in human dry eye syndrome (DES). To complement animal experiments we collected tear fluid from human dry eye patients as well as healthy control subjects. Increased tear fluid PLTP activity was observed among DES patients. In conclusion, the presence of PLTP in tear fluid appears to be essential for maintaining a healthy and functional ocular surface. Increased PLTP activity in human tear fluid in DES patients suggests an ocular surface protective role for this lipid transfer protein., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 microm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 microm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.

In the case of local anesthetic intoxication, intravenous administration of lipid-based Intralipid dispersion (Fresenius Kabi) can be used for the entrapment of hydrophobic drugs. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically trap harmful substances. In this study liposome electrokinetic capillary chromatography (LEKC) was used to study the interactions between local anesthetics and Intralipid or liposome dispersions. Intralipid dispersion and extruded liposomes with different concentrations of 1-palmitoyl-2-oleyl-sn-glycerophosphatidylcholine (POPC), phosphatidylglycerol, cardiolipin, cholesterol, oleic acid, and linoleic acid were used as a pseudostationary phase in LEKC and their interactions with lidocaine, prilocaine, and bupivacaine were studied. POPC liposomes containing 1mol% of palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine as a fluorescent marker were used for the first time in LEKC connected with laser-induced fluorescent detection in order to calculate the retention factor for anesthetics.

Fusidic acid (FA) is a potent antibiotic and blocks the protein synthesis by binding to elongation factor G (EF-G) directly. Here we hypothesized that the antibiotic activity of FA would be potentiated by several orders of magnitude if both FA and EF-G would be residing in the lipid membranes and, hence, the probability of interaction would transform from three-dimensional to two-dimensional. Such detailed information could lead to more effective therapeutic interventions if they are understood on a molecular level. Interactions between FA and various lipid membranes composed of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (Chol) were studied by capillary electrochromatography (CEC). The influence of the lipid vesicle size--sonicated liposomes and liposomes extruded through 30-, 50-, and 100-nm filters--on the packing of vesicles on the silica capillary surface was investigated by CEC and dissipative quartz crystal microbalance. The CEC results evidenced that FA interacts with and resides in phospholipid membranes. Likewise, monolayer, asymmetrical flow field flow fractionation, and CEC studies confirmed that EF-G is hydrophobic and incorporated into POPC and POPC/Chol membranes. Including EF-G in phospholipid vesicles did not improve the binding of FA to the membranes.

Objective: To evaluate visual performance after unilateral photorefractive keratectomy (PRK) performed for >/=2.75-diopter (D) myopic anisometropia., Design: Prospective observational case series., Participants: Eleven patients exhibiting >/=2.75-D myopic anisometropia appropriate for PRK., Methods: Photorefractive keratectomy was performed on 11 eyes of 11 patients for myopic anisometropia ranging from -2.75 to -9.00 D. Best spectacle-corrected visual acuity (VA), contrast sensitivity (CS), and pattern visual evoked potentials (pVEPs) were measured preoperatively and at 3 and 6 months., Main Outcome Measures: Best spectacle-corrected VA; pVEPs; and CS, measured as the area under the CS curve., Results: Best spectacle-corrected VA increased nonsignificantly 6 months after PRK. A tendency towards decreased latency time of pVEPs for both eyes was observed after PRK. A significant increase in CS occurred after PRK in the operated eye at 6 months, but a tendency toward decreased CS appeared in the unoperated eye at 3 months. Preoperative CS levels, however, were reached at 6 months., Conclusions: The adult central visual system is sensitive to visual deprivation caused by anisometropia. Imbalance between eyes may improve visual performance in the more emmetropic eye. Anisometropia appears to reduce visual performance in the more myopic eye, but this can probably be reversed by refractive correction.