Rapid quantification of free cholesterol in tears using direct insertion/electron ionization-mass spectrometry.

PURPOSE: To establish a simple and rapid analytical method, based on direct insertion/electron ionization-mass spectrometry (DI/EI-MS), for measuring free cholesterol in tears from humans and rabbits. METHODS: A stable-isotope dilution protocol employing DI/EI-MS in selected ion monitoring mode was developed and validated. It was used to quantify the free cholesterol content in human and rabbit tear extracts. Tears were collected from adult humans (n = 15) and rabbits (n = 10) and lipids extracted. RESULTS: Screening, full-scan (m/z 40-600) DI/EI-MS analysis of crude tear extracts showed that diagnostic ions located in the mass range m/z 350 to 400 were those derived from free cholesterol, with no contribution from cholesterol esters. DI/EI-MS data acquired using selected ion monitoring (SIM) were analyzed for the abundance ratios of diagnostic ions with their stable isotope-labeled analogues arising from the D6-cholesterol internal standard. Standard curves of good linearity were produced and an on-probe limit of detection of 3 ng (at 3:1 signal to noise) and limit of quantification of 8 ng (at 10:1 signal to noise). The concentration of free cholesterol in human tears was 15 ± 6 µg/g, which was higher than in rabbit tears (10 ± 5 µg/g). CONCLUSIONS: A stable-isotope dilution DI/EI-SIM method for free cholesterol quantification without prior chromatographic separation was established. Using this method demonstrated that humans have higher free cholesterol levels in their tears than rabbits. This is in agreement with previous reports. This paper provides a rapid and reliable method to measure free cholesterol in small-volume clinical samples.

Alkaloids from the roots of Stemona aphylla.

Three known compounds, stemofoline (1), (2'S)-hydroxystemofoline (2), and (11Z)-1',2'-didehydrostemofoline (3), along with two new alkaloids, stemaphylline (4) and stemaphylline-N-oxide (5), have been isolated from a root extract of Stemona aphylla. The structures of these alkaloids were determined on the basis of their spectroscopic data. The analysis of the crude dichloromethane extract by GC-MS in the EIMS mode showed the presence of alkaloids 1-4, the alkaloid 11, and stilbostemin R (12). The crude dichloromethane extract and 4 were tested for their comparative biological activities. The results of their acetylcholinesterase (AChE) inhibitory activities showed that the crude extract had higher activity than that of 4. The insecticidal properties of the crude extract and 4, using a topical application, showed that 4 had an activity similar to the positive control, methomyl, whereas the crude extract had much lower activity. Their antimicrobial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas auruginosa ATCC 27853, and Candida albicans ATCC 90028 was weak (MIC 62.5-125 microg/mL, MBC 125-250 microg/mL, MFC 125 microg/mL) but much higher than that of the crude extract.

Care regimen and lens material influence on silicone hydrogel contact lens deposition.

PURPOSE: To quantitatively detect proteins and cholesterol extracted from worn silicone hydrogel contact lenses and determine the effect of various lens care solutions on deposit accumulation. METHODS: Contact lenses, made from different polymers and worn on a daily wear schedule with different lens care solutions, were collected. Lipid and protein deposits were extracted by methanol:chloroform (1:1, v/v) and protein extraction solution (containing urea and surfactant), respectively. Lipid extracts were separated and cholesterol quantified using thin layer chromatography. Protein extracts were quantified using standard techniques. RESULTS: Among all lenses tested, Balafilcon A lenses exhibited greatest extracted cholesterol (4.1 to 8.2 microg/lens) and total protein (5.4 to 23.2 microg/lens). AQuify was the most effective solution in reducing extracted deposits, especially extracted protein, from Balafilcon A lenses. AQuify and Opti-Free RepleniSH solutions were most effective in reducing extracted cholesterol from Senofilcon A and Galyfilcon A lenses, respectively. Use of Opti-Free Express solution resulted in more extracted protein from Lotrafilcon B lenses than use of other solutions. Generally, Lotrafilcon B, Senofilcon A, and Galyfilcon A lenses accumulated relatively low amount of proteins. Lotrafilcon B lenses accumulated the least amount of cholesterol deposit among all lenses tested regardless of solution used. CONCLUSIONS: Lens polymer (possibly associated with surface characteristics) is a prominent factor affecting lipid and protein accumulation. Within a lens polymer type, lens care solutions exhibit varying effectiveness in reducing protein and lipid accumulation.

Human lens lipids differ markedly from those of commonly used experimental animals.

Electrospray ionisation tandem mass spectrometry has allowed the unambiguous identification and quantification of individual lens phospholipids in human and six animal models. Using this approach ca. 100 unique phospholipids have been characterised. Parallel analysis of the same lens extracts by a novel direct-insertion electron-ionization technique found the cholesterol content of human lenses to be significantly higher (ca. 6 times) than lenses from the other animals. The most abundant phospholipids in all the lenses examined were choline-containing phospholipids. In rat, mouse, sheep, cow, pig and chicken, these were present largely as phosphatidylcholines, in contrast 66% of the total phospholipid in Homo sapiens was sphingomyelin, with the most abundant being dihydrosphingomyelins, in particular SM(d18:0/16:0) and SM(d18:0/24:1). The abundant glycerophospholipids within human lenses were found to be predominantly phosphatidylethanolamines and phosphatidylserines with surprisingly high concentrations of ether-linked alkyl chains identified in both classes. This study is the first to identify the phospholipid class (head-group) and assign the constituent fatty acid(s) for each lipid molecule and to quantify individual lens phospholipids using internal standards. These data clearly indicate marked differences in the membrane lipid composition of the human lens compared to commonly used animal models and thus predict a significant variation in the membrane properties of human lens fibre cells compared to those of other animals.