Thomas Glonek

Tear film lipid layer thickness as a function of blinking.

Alterations in the tear film lipid layer as a function of blinking were investigated using a custom-designed specular reflection monitoring system. The tear film lipid layer of 104 subjects under conditions of normal (“baseline”) blinking and “forceful” blinking was quantitated on the basis of specific interference colors. Deliberate, forceful blinking was found to significantly increase the lipid layer thickness (LLT) of the tear film. The magnitude of increase was found to be correlated with the baseline LLT values; individuals with baseline LLT values of 75-150 nm demonstrated a mean increase in LLT of 33 nm following forceful blinking, whereas subjects with baseline LLT values ≤ 60 nm experienced a mean increase of 19 nm. The difference in the magnitude of increase between the groups was highly significant (p=0.0001). The data suggest that, in addition to playing a role in the spreading of lipid across the tear film, the blinking mechanism may be important in the maintenance of the lipid layer by augmenting the expression of lipids from the meibomian glands.

P‐31 Nuclear Magnetic Resonance Analysis of Brain: The Perchloric Acid Extract Spectrum

Abstract: Perchloric acid (PCA) extracts were prepared from liquid‐N2‐frozen guinea pig brains and their organophosphate profiles examined by P‐31 nuclear magnetic resonance (NMR) spectroscopy. Thirty‐two phosphorus‐containing brain metabolites were characterized and quantitated. A distinctive feature of brain tissue metabolism relative to that of other tissues probed by P‐31 NMR is its pronounced ribose 5‐phosphate content. Comparison of brain metabolite levels following control or sublethal cyanide treatment (4 mg/kg) revealed specific cyanide‐induced changes in brain metabolism. Brains from cyanidetreated animals were characterized by a reduced phosphocreatine content and elevated α‐glycerolphosphate and inorganic orthophosphate contents relative to control. P‐31 NMR spectra of brain PCA extracts at pH 7.2 were also obtained under conditions that approximate those used for in vivo and intact tissue in vitro P‐31 spectroscopic analyses. The spectra reveal nine separate resonance bands corresponding to: sugar phosphates, principally ribose 5‐phosphate (3.7δ); inorganic orthophosphate (2.2δ); glycerol 3‐phosphorylethanolamine (0.3δ); glycerol 3‐phosphorylcholine (−0.1δ); phosphocreatine (−3.2δ); adenosine tri‐(β‐ATP) and di‐(β‐ADP) phosphate ionized end‐groups (−6.2δ); α‐ATP, α‐ADP, and nicotinamide adenine dinucleotides esterified end‐groups (−11.1δ); uridine diphosphohexose, hexose esterified end‐groups (−13.0δ); and β‐ATP ionized middle group (−21.6δ). Knowledge of the phosphatic molecules that contribute resonances to the brain P‐31 NMR spectrum as well as understanding their magnetic resonance properties is essential for the interpretation of in vivo brain spectroscopic data as well as brain extract data, since these same compounds contribute to the intact brain P‐31 spectrum.

[53] Phosphorus-31 nuclear magnetic resonance of contractile systems

Publisher Summary This chapter focuses on techniques and procedures for recording and evaluating the 31 P nuclear magnetic resonance (NMR) spectra of intact muscles and of perchloric acid extracts of muscle. At the present time, high-resolution NMR is probably the best general-purpose analytical tool available for studying the chemistry of phosphorus and its compounds. With a 31 P NMR probe in a highresolution NMR spectrometer, the analyst can determine easily, and usually within a few minutes, whether phosphorus is present at concentrations greater than 100 μM in fluid samples, characterize each type of phosphorus-containing group present, and obtain a quantitative analysis of the various species present. For a spectrometer in which a magnetic field of 23 kiloGauss is employed so that the resonance position of protons is 100 MHz, the 31 P resonance are found at 40.5 MHz; in a magnetic field 3.6 times this, the resonance will be found at 145.8 MHz.

31P Nuclear magnetic resonancepH titrations of myo-inositol hexaphosphate

With the use of 31P n.m.r. spectroscopy, the separate pKa values of each of the six phosphoric monoester groups of myo-inositol hexaphosphate were determined. The range of hydrogen-ion concentrations covered extended from that required for the phosphonium salts to that for the full dodecyl anion, and the determinations were carried out in the presence of sodium and tetrabutylammonium cations. The pKa for each phosphate grouping in the transition from the free acid forms of each group to the monoanion form of each group was determined to be: 1.1, C-2; 1.5, C-1 and C-3; 2.1, C-4 and C-6; and 1.7, C-5. In the mono- to di-anion transition, the pKa values were: 6.85, C-2; 7.60, C-5; 5.70 and 12.0, C-1 and C-3; and 10.0, C-4 and C-6. These data and the appearance of the 31P hexaphosphate n.m.r. multiplet are discussed in terms of conformations of myo-inositol hexaphosphate.

Correlation of lipid layer thickness measurements with fluorescein tear film break-up time and Schirmer's test

AbstractPurpose This study correlates measurement of lipid layer thickness (LLT) with two frequently used dry eye tests, fluorescein break-up time (FBUT) and Schirmers test with anaesthesia (STA).Methods Subjects (n=44 eyes) with symptoms of dry eye and positive results for dry eye with either FBUT or STA or both were selected. Quantification of LLT was performed by the observation of colour interference patterns in zones of specular reflection using a custom-designed instrument.Results All correlations among pairs of tests were strong and exhibited a significance of P<0.000: STA with FBUT, Pearsons correlation 0.653; STA with LLT, 0.764; FBUT with LLT, 0.751. When LLT was high, ie ≥120 nm, which occurred in 14 eyes, STA was also elevated in those eyes and FBUT was high in 13 of the 14 eyes. When LLT was low, ie ≤60, which occurred in 22 eyes, STA was below normal in 14 of the 22 eyes, and FBUT was below normal in 15 of the 22 eyes. These clinical observations paralleled the statistical findings computed from the entire data set.Conclusions The correlations demonstrated in this study support the premise (1) that measurement of LLT is a reliable test for the diagnosis of dry eye, and (2) that aqueous deficiency and lipid deficiency, as they apply to dry eye disorders, are not mutually exclusive.

Lid-wiper epitheliopathy and dry-eye symptoms in contact lens wearers

PURPOSE To evaluate whether dry-eye symptoms are associated with epitheliopathy of that portion of the upper eyelid marginal conjunctiva-the lid wiper-that wipes the ocular, or contact lens surface, during blinking. METHODS Subjects were divided into two groups based on the presence or absence of dry-eye symptoms. The lid wiper of asymptomatic (n=75) and symptomatic (n=30) soft contact lens wearers was examined, following the instillation of fluorescein and rose bengal dyes. Lid-wiper staining was graded zero to 3. RESULTS Eighty percent of the symptomatic subjects displayed lid-wiper staining compared to 13% of the asymptomatic subjects. The difference in staining between the two groups was significant (P<0.0001). Of the symptomatic subjects, 20% showed no staining; 26.6%, grade 1 staining; 36.6%, grade 2; and 16.6% showed grade 3 staining. Of the asymptomatic subjects, 87% exhibited no staining; 9%, grade 1 staining; 3%, grade 2; and 1% showed grade 3 staining. CONCLUSIONS This study describes a new clinical condition, lid-wiper epitheliopathy, an alteration of the epithelium of that portion of the marginal conjunctiva of the upper eyelid that wipes the ocular surface, diagnosed by staining with fluorescein and rose bengal dyes.

Phospholipids in Meibomian Gland Secretion

The bulk of the lipid layer overlying the aqueous portion of the precorneal tear film is composed of polar and nonpolar components. The nonpolar lipids have been the subject of numerous studies; however, the polar lipids have remained relatively uncharacterized. The polar lipids are thought to contain surfactant phospholipids that are critical to the spreading of a lipid film over the aqueous layer, by providing an interface between this layer and the nonpolar lipids. The purpose of the present study is to identify and quantitate the phospholipid complement of meibomian gland secretion which provides the tear film with phospholipids. Meibomian gland secretion was collected from rabbits and phospholipids identified and quantitated by 31P nuclear magnetic resonance spectroscopy. Ten phospholipids were detected from meibomian gland secretion: diphosphatidylglycerol, dihydrosphingomyelin, ethanolamine plasmalogen, phosphatidylethanolamine (PE), phosphatidylserine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol, alkylacylphosphatidylcholine, and phosphatidylcholine (PC). The two major phospholipids were PC and PE, together comprising nearly 60% of the total phospholipid profile. The nature and relative concentrations of the meibomian gland secretion phospholipids are congruous with a surfactant role at the aqueous-lipid interface and, considering the physical chemistry of the tear film, suggest that the phospholipids should be organized in a very flat or planar configuration.

Meibomian gland phospholipids

The content of the meibomian gland lipid exprimate is known, but little is known about the phospholipids that comprise the glandular cells. The purpose of the present study is to identify and quantitate the phospholipid complement of the meibomian gland cells that produce the lipid secretion of meibomian oil and which is vital to tear film stability. Eyelids (n = 50) were excised from rabbits, and after surgical removal of surrounding tissues, the tarsal plates with and without expressing meibomian oil were extracted and phospholipids of the plates quantified by 31P nuclear magnetic resonance (NMR). Seventeen phospholipids were quantified from tarsal plates expressed of oil and tarsal plates containing meibomian oil: alkylacylphosphatidylcholine (AAPC), dihydrosphingomyelin (DHSM), dimethylphosphatidylethanolamine, diphosphatidylglycerol (cardiolipin), ethanolamine plasmalogen (EPLAS), lysoethanolamine plasmalogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidic acid, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin (SM), sphingosylphosphorylcholine. The six zwitterionic and neutral phospholipids, DHSM, EPLAS, PE, SM, AAPC, and PC together comprise 79.5% of the total meibomian gland phospholipid profile (in meibomian oil this value is 84.2%). The zwitterionic and neutral phospholipids dominate meibomian gland phospholipid profiles. Since the meibomian gland cells undergo holocrine secretion and form the meibomian glad secretion, such a composition is consistent with the hypothesis that a chemically stable lamellar surfactant layer phospholipids bind non-polar meibomian oil to the aqueous layer of the tear film.

Effect of periocular humidity on the tear film lipid layer.

The purpose of this study was to determine the relationship between the tear film and humidity by examining whether alterations in periocular humidity influence the thickness of the tear film lipid layer. Thirteen dry eye subjects presenting with a baseline lipid layer thickness of ≤60 nm were fitted with modified swim goggles in which the right eye (OD) was exposed to conditions of high humidity and the left eye (OS) remained exposed to ambient room conditions. The lipid layer was monitored over a 60-min time course with goggles on and for an additional 60 min following goggle removal. The OD lipid layer increased significantly in thickness within 5 min of exposure to conditions of high humidity (p<0.0001), reaching a maximum increase of 66.4 nm after 15 min of goggle wear (p<0.0001). This maximum increase to a lipid layer thickness of 120.5 nm was maintained at the 30- and 60-min goggle time points. No significant change was detected OS. Following goggle removal, OD values declined but remained significantly elevated over the OS lipid layer thickness throughout the 60-min postgoggle period. Moderate to total relief of dry eye symptoms was reported during goggle wear and generally persisted at a reduced level for 1-3 h following goggle removal. Increased periocular humidity results in an increase in tear film lipid layer thickness, possibly by providing an environment that is more conducive to the spreading of meibomian lipid and its incorporation into the tear film.

Sodium-23 NMR analysis of human whole blood, erythrocytes, and plasma. Chemical shift, spin relaxation, and intracellular sodium concentration studies

Sodium-23 NMR analysis was performed on freshly obtained human whole blood, erythrocytes, and plasma. The intracellular and extracellular sodium signals were separated by adding dysprosium: tripolyphosphate to the plasma bathing the erythrocytes. Quantitation of the intracellular sodium content was easily accomplished by sodium NMR and was shown to agree well with the values obtained by flame photometry. T1 and T2 relaxation studies demonstrated that the sodium in human plasma and within human erythrocytes is substantially different in its physical characteristics than sodium in aqueous solution, and that some fraction of the plasma and erythrocyte sodium is relatively immobilized. Sodium NMR would appear therefore to be a useful method for studying sodium biology in inherited and acquired human diseases.