Randomized comparison of in vivo performance of TearLab® and I-PEN® osmometry in normal dogs.

OBJECTIVE: To compare tear film (TF) osmolarity measured using TearLab® and I-PEN® osmometers in the same dogs without any ocular surface disease. ANIMAL STUDIED: Fifty-two dogs (98 eyes) of different breeds were evaluated. PROCEDURES: Tear film (TF) osmolarity was evaluated at 2-min intervals. The test was randomly determined, and single measurements were performed using each osmometer. Subsequently, complete ophthalmologic examinations were performed based on Schirmer tear test-1 (STT-1) analysis, tear film breakup time (TFBUT), and slit-lamp biomicroscopy. For each osmometer, the mean ± standard deviation of the TF osmolarity was calculated, and a paired Student's t-test was used to compare the values obtained. Pearson correlation analysis was performed to assess the association between osmolarity and other values such as STT-1, TFBUT, and age. RESULTS: Tear film osmolarity determined using TearLab® (340.42 ± 15.87 mOsm/L) and I-PEN® (321.58 ± 17.39 mOsm/L) were significantly different (p < .001). However, statistical significance could not be confirmed between osmolarity and other values, such as STT-1, TFBUT, and age. CONCLUSIONS: In dogs, the TF osmolarity values obtained using TearLab® tend to be higher than those obtained using I-PEN®, contrary to that observed in humans. These findings can serve as a reference for establishing normal values for each osmometer for clinical use in measuring TF osmolarity in dogs.

Determination of specific and non-specific protein-protein interactions for beta-lactoglobulin by analytical ultracentrifugation and membrane osmometry experiments.

Protein-protein interactions are essential for the understanding of biological processes. Specific protein aggregation is an important aspect for many biological systems. In particular, electrostatic interactions play the key role for protein-protein interactions, as many amino acids have pH-dependent charge states. Moreover, protein dissociation is directly related to the solution pH, ionic strength, temperature and protein concentration. The subtle interplay between different specific and non-specific interactions is demonstrated for beta-lactoglobulin (BLG) with a focus on low salt concentrations, thus mimicking technically relevant processing conditions. BLG is a well-characterized model system, proven to attain its monomer-dimer equilibrium strongly dependent upon the pH of the solution. In this manuscript, we present a unique combination of analytical ultracentrifugation and membrane osmometry experiments, which quantifies specific and non-specific interactions, i.e. in terms of the dimer dissociation constants and the second osmotic virial coefficient, at pH 3 and 7 and sodium chloride concentrations of 10 mM and 100 mM. This provides direct insight to protein-protein interactions for a system with a concentration-dependent monomer-dimer equilibrium. Moreover, using a coarse-grained extended DLVO model in combination with molecular dynamics simulations, we quantify non-specific monomer-monomer, monomer-dimer and dimer-dimer interactions as well as the binding free energy of BLG dimerization from theoretical calculations. The experimentally determined interactions are shown to be mainly governed by electrostatic interactions and further agree with free energy calculations. Our experimental protocol aims to determine non-specific and specific interactions for a dynamically interacting system and provides an understanding of protein-protein interactions for BLG at low salt concentrations.

Comparison of measured and calculated osmolality levels.

BACKGROUND: Serum osmolality levels are measured to determine acid-base and electrolyte imbalance in serum. In cases where measurement is not possible, the serum osmolality value can be calculated by various calculation methods. In this study, we compared the Worthley osmolality calculation method which is used most frequently mentioned in literature and the measurements made with vapor pressure osmometer used in our laboratory. We compared whether there was a difference between the results obtained by measurement and calculation method in different age groups. METHODS: 221 serum samples of patients who were admitted to the Eskisehir Osmangazi University Hospital Biochemistry Laboratory between December 2016 and May 2018 were included in this study. Glucose, blood urea nitrogen and sodium values were recorded to determine the calculated osmolality values of the patients. RESULTS: There was a statistically significant difference between the measured osmolality values and the calculated osmolality values of the patients (p < 0.001). When compared according to age groups, there was a significant difference between calculated osmolality values (p = 0.006), but there was no difference in measured osmolality values (p = 0.787) in different age groups. It has been observed that this difference in the calculated osmolality values between the age groups is derived from the adult group (18-65, p < 0.001). CONCLUSION: Our results showed that it is not reliable to calculate serum osmolality values, especially in the adult age group. According to our results the calculated osmolality values are higher than our measured osmolality values.

Electrochemical Investigation of the Interaction between Catecholamines and ATP.

The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

Calculated Versus Measured Urine Osmolarity: Accuracy of Estimated Urine Density.

BACKGROUND: Urine osmolarity (OsmU) is the gold standard for the evaluation of the kidney's urine concentration capacity; nevertheless, urinary density (UD) is often used as a surrogate for its estimation. OBJECTIVE: The objective of this study was to analyze the accuracy of UD in estimating OsmU. MATERIALS AND METHODS: A transversal study including patients with simultaneous determination of UD measured with refractometry and OsmU measured by osmometer (OsmUm). We multiplied the last two digits of the UD by 35, 30, 32, 33.5, and 40 to estimate OsmU; the estimates were considered precise if the value was ± 30 mOsm/kg from the OsmUm. A Bland-Altman analysis was conducted. RESULTS: Among 205 patients, there was no difference between OsmUm and the estimated form when using a factor of 33.5 (p = 0.578). When analyzing by the absence or presence of proteinuria and/or glycosuria, there were no differences when using the factors 35 (p = 0.844) and 32 with adjusted UD (p = 0.898). In the linear correlation analysis, values for Pearson's r = 0.788 and r2 = 0.621 were obtained (p < 0.001). The areas under the curve obtained by the receiver operating characteristics curves to estimate urine osmolarity values < 100 and > 600 mOsm/kg were > 0.90. CONCLUSION: The estimation of the OsmU from UD showed adequate performance. If an osmometer is unavailable, we recommend using the factor 35 for clean samples and 32 with adjusted UD for samples with proteinuria and/or glycosuria.

How Osmolytes Counteract Pressure Denaturation on a Molecular Scale.

Life in the deep sea exposes enzymes to high hydrostatic pressure, which decreases their stability. For survival, deep sea organisms tend to accumulate various osmolytes, most notably trimethylamine N-oxide used by fish, to counteract pressure denaturation. However, exactly how these osmolytes work remains unclear. Here, a rigorous statistical thermodynamics approach is used to clarify the mechanism of osmoprotection. It is shown that the weak, nonspecific, and dynamic interactions of water and osmolytes with proteins can be characterized only statistically, and that the competition between protein-osmolyte and protein-water interactions is crucial in determining conformational stability. Osmoprotection is driven by a stronger exclusion of osmolytes from the denatured protein than from the native conformation, and water distribution has no significant effect on these changes at low osmolyte concentrations.

[New approaches to the treatment of keratoconjunctivitis sicca]. / Novye podkhody k lecheniiu sukhogo keratokon''iunktivita.

A new method has been developed for the treatment of severe forms of keratoconjunctivitis sicca (KCS) that involves the use of an original cyclosporine A (CyA) saturated soft contact lens (SCL) together with preservative-free artificial tears therapy. AIM: to evaluate the effectiveness of the newly developed treatment for KCS based on the use of medical SCL saturated with 0.05% CyA. MATERIAL AND METHODS: The patients (43 men, 60 eyes) with severe KCS were divided into 2 groups. Group 1 included 21 patients (30 eyes), who received artificial tears and wore 0.05% CyA-saturated silicone-hydrogel SCLs. Group 2 included 22 patients (30 eyes), who wore unsaturated original SCLs and received CyA instillations 2 times daily and, also, artificial tears. Apart from a standard ophthalmic examination, the assessment included Schirmer's test, Norn's test, vital eye stain tests, tear osmometry, laser confocal tomography of the cornea, optical coherence tomography of the anterior segment with meniscometry, impression cytology of the conjunctiva, tear pH measurement, plating of the content of the conjunctival cavity, measurement of the width of the palpebral fissure, and calculation of the ocular surface disease index. Treatment results were followed up at 1, 3, 6, and 12 months. RESULTS: The use of 0.05% CyA-saturated SCLs allows to halve treatment time for patients with severe KSC (down to 1 week - 1 month) as compared to unsaturated original SCLs in combination with 0.05% CyA instillations and to reduce it 5 times as compared to 0.05% CyA instillations only. CONCLUSION: The new method of KSC treatment that involves the use of medical SCL of original design (ensures even distribution of 0.05% CyA across the ocular surface) and preservative-free artificial tears has demonstrated high therapeutic effectiveness as compared to existing methods.

Lack of Agreement among Electrical Impedance and Freezing-Point Osmometers.

PURPOSE: To assess the interchangeability of tear osmolarity measurements between electrical impedance and freezing-point depression osmometers and to analyze inter-eye tear osmolarity variability measured with these osmometers in healthy subjects. METHODS: Tear osmolarity was measured using the TearLab osmometer (OcuSense Inc., San Diego, CA) and the Fiske 210 microsample osmometer (Advanced Instruments Inc., Norwood, MA). We randomly selected one eye in 50 subjects (29 women, 21 men; mean age, 33.16 ± 6.11 years) to analyze whether osmolarity measurements by these osmometers were interchangeable. Both eyes of 25 patients (15 women, 10 men; mean age, 34.32 ± 6.37 years) were included to analyze inter-eye osmolarity variability. RESULTS: The mean tear osmolarity values measured with the TearLab osmometer were higher (305.22 ± 16.06 mOsm/L) than those with the Fiske 210 osmometer (293.40 ± 12.22 mOsm/L), with the intraclass correlation coefficient being 0.23 (p = 0.051). A Bland-Altman plot showed that the systems were not interchangeable because there was a systematic difference, with the limits of agreement being -17.93 to 41.57 mOsm/L. There were no statistically significant differences (p = 0.5006 and p = 0.6533, respectively) between an individual's eyes measured with either osmometer. CONCLUSIONS: Because the TearLab tear osmolarity measurements were higher than those of the Fiske 210 measurements and the limits of agreement were too wide, the two osmolarity values cannot be used interchangeably. In healthy subjects, there is no difference in tear osmolarity between right and left eyes of the same individual measured with both instruments.

Calculating osmotic pressure of glucose solutions according to ASOG model and measuring it with air humidity osmometry.

The osmotic pressure of glucose solution at a wide concentration range was calculated using ASOG model and experimentally determined by our newly reported air humidity osmometry. The measurements from air humidity osmometry were compared with the well-established freezing point osmometry and ASOG model calculations at low concentrations and with only ASOG model calculations at high concentrations where no standard experimental method could serve as a reference for comparison. Results indicate that air humidity osmometry measurements are comparable to ASOG model calculations at a wide concentration range, while at low concentrations freezing point osmometry measurements provide better comparability with ASOG model calculations.

Its preferential interactions with biopolymers account for diverse observed effects of trehalose.

Biopolymer homeostasis underlies the health of organisms, and protective osmolytes have emerged as one strategy used by Nature to preserve biopolymer homeostasis. However, a great deal remains unknown about the mechanism of action of osmolytes. Trehalose, as a prominent example, stabilizes proteins against denaturation by extreme temperature and denaturants, preserves membrane integrity upon freezing or in dry conditions, inhibits polyQ-mediated protein aggregation, and suppresses the aggregation of denatured proteins. The underlying thermodynamic mechanisms of such diverse effects of trehalose remain unclear or controversial. In this study, we applied the surface-additive method developed in the Record laboratory to attack this issue. We characterized the key features of trehalose-biopolymer preferential interactions and found that trehalose has strong unfavorable interactions with aliphatic carbon and significant favorable interactions with amide/anionic oxygen. This dissection has allowed us to elucidate the diverse effects of trehalose and to identify the crucial functional group(s) responsible for its effects. With (semi)quantitative thermodynamic analysis, we discovered that 1) the unfavorable interaction of trehalose with hydrophobic surfaces is the dominant factor in its effect on protein stability, 2) the favorable interaction of trehalose with polar amides enables it to inhibit polyQ-mediated protein aggregation and the aggregation of denatured protein in general, and 3) the favorable interaction of trehalose with phosphate oxygens, together with its unfavorable interaction with aliphatic carbons, enables trehalose to preserve membrane integrity in aqueous solution. These results provide a basis for a full understanding of the role of trehalose in biopolymer homeostasis and the reason behind its evolutionary selection as an osmolyte, as well as for a better application of trehalose as a chemical chaperone.

Apoplastic water fraction and rehydration techniques introduce significant errors in measurements of relative water content and osmotic potential in plant leaves.

Relative water content (RWC) and the osmotic potential (π) of plant leaves are important plant traits that can be used to assess drought tolerance or adaptation of plants. We estimated the magnitude of errors that are introduced by dilution of π from apoplastic water in osmometry methods and the errors that occur during rehydration of leaves for RWC and π in 14 different plant species from trees, grasses and herbs. Our data indicate that rehydration technique and length of rehydration can introduce significant errors in both RWC and π. Leaves from all species were fully turgid after 1-3 h of rehydration and increasing the rehydration time resulted in a significant underprediction of RWC. Standing rehydration via the petiole introduced the least errors while rehydration via floating disks and submerging leaves for rehydration led to a greater underprediction of RWC. The same effect was also observed for π. The π values following standing rehydration could be corrected by applying a dilution factor from apoplastic water dilution using an osmometric method but not by using apoplastic water fraction (AWF) from pressure volume (PV) curves. The apoplastic water dilution error was between 5 and 18%, while the two other rehydration methods introduced much greater errors. We recommend the use of the standing rehydration method because (1) the correct rehydration time can be evaluated by measuring water potential, (2) overhydration effects were smallest, and (3) π can be accurately corrected by using osmometric methods to estimate apoplastic water dilution.

Accuracy of a Freezing Point Depression Technique Osmometer.

PURPOSE: The purpose of this study was to examine the precision and accuracy of the Fiske 110 Osmolarity System under different protocols to determine the possible applications of this device in tear film research and clinical practice. METHODS: Three separate studies were performed. In the first, Fiske 110 measurements were made on undiluted and diluted (1:1, 1:4, and 1:9 dilutions) standard samples of different osmolarity values: 50, 290, and 850 mOsm/kg and 297 and 338 mOsm/L. In the second study, measurements were made on different types of contact lens care solutions. Finally, in an agreement study, measurements were made in two sets of 60 subjects to compare TearLab versus Fiske 110 (using both 2- and 4-µL tear sample). RESULTS: Although osmolarity measurements for undiluted solutions differed statistically from reference standard values, all biases were in the tolerance range proposed by the manufacturer except for the 850-mOsm/kg solution. No significant differences from reference osmolarity values were observed for the 1:1 and 1:4 diluted 297- and 338-mOsm/L H2O solutions, respectively, although all diluted solutions showed a possible bias out of the range provided. Osmolarities for the soft contact lens solutions fell within the range 293 to 309 mOsm/kg with the exception of Opti-Free Express (225 mOsm/kg). In the agreement study, significant differences were observed between measurements obtained using the TearLab and both Fiske 110 procedures, although the Fiske 110 (4 µL) procedure was closer to the TearLab than the Fiske (2 µL) procedure. CONCLUSIONS: For undiluted solutions, the Fiske 110 shows good performance, making it a useful device for osmolarity measurements in lens care solutions or eye drops. A worse performance was observed for more diluted standard solution samples. When testing diluted samples, performance was acceptable for osmolarity values close to tear values.

The structure of 3-(diethylborylethynyl)pyridine: a nonplanarly arranged cyclic trimer.

3-(Diethylborylethynyl)pyridines 2 assemble into a cyclic trimer stabilized via intermolecular boron­nitrogen coordination bonds both in solution and in the crystalline state. The outstanding structural features of the methoxy derivative 2b in the crystalline state are that (1) two pyridine rings (P1 and P2) of the cyclic trimer of 2b are almost coplanar, and the third pyridine ring (P3) is largely bent away from P1 and P2, and (2) P3 of the cyclic trimer stacks in a face-to-face fashion with one of the pyridine rings (P3') of an adjacent cyclic trimer. The crystallographic study revealed that the conformation of the cyclic trimer is flexible enough to be affected by the crystal packing.

Determining osmotic pressure of drug solutions by air humidity in equilibrium method.

OBJECTIVE: To establish a new osmotic pressure measuring method with a wide measuring range. METHOD: The osmotic pressure of drug solutions is determined by measuring the relative air humidity in equilibrium with the solution. The freezing point osmometry is used as a control. RESULTS: The data obtained by the proposed method are comparable to those by the control method, and the measuring range of the proposed method is significantly wider than that of the control method. CONCLUSION: The proposed method is performed in an isothermal and equilibrium state, so it overcomes the defects of the freezing point and dew point osmometries which result from the heterothermal process in the measurement, and therefore is not limited to diluted solutions.

Calculating osmotic pressure of xylitol solutions from molality according to UNIFAC model and measuring it with air humidity osmometry.

The osmotic pressure of xylitol solution at a wide concentration range was calculated according to the UNIFAC model and experimentally determined by our newly reported air humidity osmometry. The measurements from air humidity osmometry were compared with UNIFAC model calculations from dilute to saturated solution. Results indicate that air humidity osmometry measurements are comparable to UNIFAC model calculations at a wide concentration range by two one-sided test and multiple testing corrections. The air humidity osmometry is applicable to measure the osmotic pressure and the osmotic pressure can be calculated from the concentration.

Cross-validation of the osmotic pressure based on Pitzer model with air humidity osmometry at high concentration of ammonium sulfate solutions.

The osmotic pressure of ammonium sulfate solutions has been measured by the well-established freezing point osmometry in dilute solutions and we recently reported air humidity osmometry in a much wider range of concentration. Air humidity osmometry cross-validated the theoretical calculations of osmotic pressure based on the Pitzer model at high concentrations by two one-sided test (TOST) of equivalence with multiple testing corrections, where no other experimental method could serve as a reference for comparison. Although more strict equivalence criteria were established between the measurements of freezing point osmometry and the calculations based on the Pitzer model at low concentration, air humidity osmometry is the only currently available osmometry applicable to high concentration, serves as an economic addition to standard osmometry.

Inorganic ion composition in Tardigrada: cryptobionts contain a large fraction of unidentified organic solutes.

Many species of tardigrades are known to tolerate extreme environmental stress, yet detailed knowledge of the mechanisms underlying the remarkable adaptations of tardigrades is still lacking, as are answers to many questions regarding their basic biology. Here, we present data on the inorganic ion composition and total osmotic concentration of five different species of tardigrades (Echiniscus testudo, Milnesium tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus crispae) using high-performance liquid chromatography and nanoliter osmometry. Quantification of the ionic content indicates that Na(+) and Cl(-) are the principal inorganic ions in tardigrade fluids, albeit other ions, i.e. K(+), NH4(+), Ca(2+), Mg(2+), F(-), SO4(2-) and PO4(3-) were also detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor compared with that of the external medium (Na(+), ×70-800; K(+), ×20-90; Ca(2+) and Mg(2+), ×30-200; F(-), ×160-1040, Cl(-), ×20-50; PO4(3-), ×700-2800; SO4(2-), ×30-150). In contrast, in the marine species H. crispae, Na(+), Cl(-) and SO4(2-) are almost in ionic equilibrium with (brackish) salt water, while K(+), Ca(2+), Mg(2+) and F(-) are only slightly concentrated (×2-10). An anion deficit of ~120 mEq l(-1) in M. tardigradum and H. crispae indicates the presence of unidentified ionic components in these species. Body fluid osmolality ranges from 361±49 mOsm kg(-1) in R. coronifer to 961±43 mOsm kg(-1) in H. crispae. Concentrations of most inorganic ions are largely identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not lose large quantities of inorganic ions during dehydration. The large osmotic and ionic gradients maintained by both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in Tardigrada. Moreover, our data indicate that cryptobiotic tardigrades contain a large fraction of unidentified organic osmolytes, the identification of which is expected to provide increased insight into the phenomenon of cryptobiosis.

In situ osmometry: validation and effect of sample collection technique.

PURPOSE: To compare tear film osmolarity measurements between in situ and vapor pressure osmometers. Repeatability of in situ measurements and the effect of sample collection techniques on tear film osmolarity were also evaluated. METHODS: Osmolarity was measured in one randomly determined eye of 52 healthy participants using the in situ (TearLab Corporation, San Diego, CA) and the vapor pressure (Vapro 5520; Wescor, Inc., Logan, UT) osmometers. In a subset of 20 participants, tear osmolarity was measured twice on-eye with the in situ osmometer and was additionally determined on a sample of nonstimulated collected tears (3 µL) with both instruments. RESULTS: Mean (SD) tear film osmolarity with the in situ osmometer was 299.2 (10.3) mOsmol/L compared with 298.4 (10) mmol/kg with the vapor pressure osmometer, which correlated moderately (r = 0.5, P < 0.05). Limits of agreement between the two instruments were -19.7 to +20.5 mOsmol/L. Using collected tears, measurements with the vapor pressure osmometer were marginally higher (mean [SD], 303.0 [11.0] vs 299.3 [8.0] mOsmol/L; P > 0.05) but correlated well with those using the in situ osmometer (r = 0.9, P < 0.05). The mean (SD) osmolarity of on-eye tears was 5.0 (6.6) mOsmol/L higher than that of collected tears, when both measurements were conducted with the in situ osmometer. This was a consistent effect because the measurements correlated well (r = 0.65, P < 0.05).The in situ osmometer showed good repeatability with a coefficient of repeatability of 9.4 mOsmol/L (r = 0.8, P < 0.05). CONCLUSIONS: Correlation between the two instruments was better when compared on collected tear samples. Tear film osmolarity measurement is influenced by the sample collection technique with the osmolarity of on-eye tears being higher than that of collected tears. This highlights the importance of measuring tear film osmolarity directly on-eye. The in situ osmometer has good repeatability for conducting this measurement.

A needle micro-osmometer for determination of glycosaminoglycan concentration in excised nucleus pulposus tissue.

PURPOSE: Aggrecan is one of the major macromolecular components of the intervertebral disc (IVD) and its loss is an early sign of degeneration. Restoration of aggrecan, and hence of biomechanical properties, is a major objective of biological therapies. At present, assessment of aggrecan concentration via its glycosaminoglycan (GAG) content is accomplished using biochemical and histological methods which require sacrifice of tissue. A minimally invasive method for assessing GAG, and hence aggrecan, which can avoid destruction of tissue, would be of benefit. METHODS: We have developed a needle micro-osmometer that is capable of measuring flux of saline into excised human nucleus pulposus (NP) tissue. Using the isotropic osmotic stress technique to assess the swelling pressure of the excised NP tissue and assuming negligible collagen tensile stress, we were able to relate the flux to the tissue fixed charge density (FCD). GAG concentration is evaluated from its FCD via the radioactive tracer technique. Samples representing different ages (28-59 years) and degeneration grades (1-4) were analyzed. RESULTS: The flux is controlled by both the osmotic pressure difference across the probe's semi-permeable membrane and by the tissue permeability. A linear correlation was found between flux and the tissue FCD. The equation describing the linear fit is FCD/(total tissue hydration) = 1.97 × 10(-4) + 8283 × flux (R = 0.836, p < 10(-4)). Thus, by measuring saline flux, the concentration of GAG can be determined. CONCLUSIONS: Micro-osmometry provides a reliable and minimally invasive tool for assessing GAG content in excised NP tissue. This method may be usefully applied in tissue engineering applications. It may also be useful for in vivo measurements if the question of the degenerative effect of needle puncture can be overcome.