Long-chain fatty acids and alcohols from gerbil meibomian lipids.

Capillary gas chromatography-mass spectrometry was used to identify constituent fatty acids, alcohols and steroids from gerbil meibomian glands. Over 80 compounds representing about 90% of the total fraction were identified. The major steroid was cholesterol accompanied by a lower concentration of 3 beta-hydroxy-5 alpha-cholestane. Fatty acids with chain lengths from 12 to 27 carbon atoms were present; they had predominantly straight, iso or anteiso chains with major concentrations in the C15-C18 and C25-C27 regions. Unsaturated acids had mainly 16 and 18 carbon atoms. The fatty alcohols were mainly branched-chain with the majority of compounds having chain lengths of 25-27 carbon atoms. Several alcohols, both branched and unsaturated, were found with chains of up to 33 carbon atoms long. The profile was similar to that found earlier in other species, but with lower concentrations of mono-unsaturated compounds than were found in rats and humans. Di-hydroxy compounds, on the other hand, tended to be more abundant although still of low relative concentration.

The role of wax and sterol esters of meibomian secretions in chronic blepharitis.

We analyzed the fatty wax esters and sterol esters found in the expressed lipid secretions of six patients from each of six clinical groups of chronic blepharitis, plus eight normal controls. Using gas liquid chromatography (GLC), 12 peaks corresponding to equivalent chain lengths (ECL) of 33.6, 35.4, 36.1, 37.3, 38.2, 39.2, 40.1, 41.2, 42.1, 43.2, 44.9 and 45.7 were found in the fatty wax esters and five peaks corresponding to ECL 19.1, 20.0, 21.1, 22.0 and 23.2 were found in the sterol esters. The clinical groups showed significant differences in several of these components. Sterol and wax esters represent the largest fraction of the total meibomian lipid secretion. The finding that the blepharitic groups exhibit biochemical differences in the distribution of these esters indicates that the esters may play a role in the disease process, perhaps by providing a preferential substrate for normal flora which we have shown to have lipolytic capabilities. The resultant de-esterification could alter the tear film and contribute to ocular irritation. In addition, the differences seen in the composition of the ester fraction of the meibomian secretion could lead to differences in melting point, which, in turn, could affect viscosity and surface tension of the tear film, leading to changes in breakup time and to secretion stagnation within glands.

Eyelid pigment implantation: early and late histopathology.

Full-thickness sections of upper and lower eyelids were obtained from patients who had eyelid pigment implantation performed by one surgeon. These four patients represented eyelid pigment implantation at varying postoperative stages ranging from 6 months to 4 years. Light microscopic evaluation revealed pigment within dermis and superficial orbicularis muscle. Light and electron microscopic evaluation revealed the vast majority of the pigment to be located intracellularly, primarily within macrophages. No foreign-body reaction was seen around the implanted material. Electron probe analysis of the pigment showed only the presence of iron. Analysis of the unused pigment revealed small amounts of silica and magnesium. These substances could not be identified by electron probe analysis in the eyelid tissues.

Identification by combined gas chromatography-mass spectrometry of constituent long-chain fatty acids and alcohols from the meibomian glands of the rat and a comparison with human meibomian lipids.

Constituent long-chain fatty acids and alcohols from the meibomian secretions of the rat were examined as trimethylsilyl (TMS) and methyl ester-TMS derivatives by capillary gas chromatography and by combined gas chromatography-mass spectrometry. The positions of double bonds and methyl branch points were determined by the mass spectra of picolinyl esters and nicotinates for long-chain fatty acids and alcohols, respectively. Fatty acids had chain lengths from C12 to C34 and were of the straight-chain iso, anteiso and monounsaturated types. The unsaturated acids had double bonds in the omega-7 and omega-9 positions. The alcohols had corresponding structures. In common with the constituent acids and alcohols of other meibomian secretions, the chain lengths of the constituents showed a biphasic distribution with maxima around C16-C18 and C25-C27. The profile was qualitatively similar to that obtained from human meibomian secretion but with some differences in the relative proportions of certain acids and alcohols.

The di- and triesters of the lipids of steer and human meibomian glands.

Three groups of diesters have been isolated and identified in the lipids of steer meibomian glands. The first group, designated as alpha Type I, with the abbreviated formula FA-alpha OHFA-FAlc, consisted of alpha-hydroxy fatty acids esterified to fatty acids and fatty alcohols in the approximate molar ratio 1:1:1. The second group, designated as omega Type I-St, with the abbreviated formula FA-omega OHFA-St, consisted of omega-hydroxy fatty acids esterified to fatty acids and sterols in the approximate molar ratio 1:1:1. The third group, designated as alpha, omega Type II, with the abbreviated formula FA-alpha, omega diol-FA, consisted of alpha, omega-diols esterified to 2 moles of fatty acids. The sum of the different diesters comprised about 9% of total steer meibomian lipids. Capillary GLC of the fatty acids of alpha Type I diesters showed the fatty acids to be a family with a two-cluster profile, one at C12 to C20 and the other at C21 to C31, with anteiso chains predominating. Fatty acids from omega Type I-St and alpha, omega Type II diesters gave mainly a one-cluster profile in the short chain region with prominent anteiso and C18:1 peaks. Fatty alcohols of alpha Type I diesters were mainly long chain, C23 to C30, with anteiso chains predominating, while the alpha-hydroxy fatty acids were short chain C13 to C18 acids with C16 predominating. The sterols in diesters omega Type I-St were cholesterol (approximately 60%), delta 7 cholestenol (approximately 35%) and an unidentified compound (approximately 5%) with a GLC retention time slightly longer than delta 7 cholestenol on SE-30 phase. The omega-hydroxy fatty acids and alpha, omega-diols both were of exceedingly long chain lengths, C29-C38, and showed similar GLC profiles. Two types of triesters comprising approximately 1% of total steer meibomian lipids have been isolated but incompletely characterized. In terms of molar ratios, one group of triesters gave fatty acids:omega-hydroxy fatty acids:alpha-hydroxy fatty acids:sterols + fatty alcohols as approximately 1:1:1:1. The other contained fatty acids, alpha-hydroxy fatty acids and alpha, omega-diols in what appears to be a complex mixture of several triesters. Diesters omega Type I and alpha, omega Type II also were found in human meibum. Hitherto these two diesters have not been found in any animal tissue.

Identification of meibomian gland lipids by gas chromatography-mass spectrometry: application to the meibomian lipids of the mouse.

Methods are described for the structural determination of the constituent alcohols, steroids and fatty acids from meibomian gland esters. The compounds, obtained from hydrolysed extracts, were separated and quantified as trimethylsilyl and methyl ester-trimethylsilyl derivatives by fused-silica capillary column gas chromatography. Structural information relating to the position of chain branching and the position of double bonds in the aliphatic chains of the acids and alcohols was obtained from the mass spectra of a number of other derivatives. Thus pyrrolidide-trimethylsilyl and picolinyl ester-trimethylsilyl derivatives provided information on both branching and unsaturation. The double-bond position in unsaturated fatty acids was also examined by use of trimethylsilyl derivatives of the derived glycols. The positions of chain branching of the alcohols were determined by the spectra of their acetate and nicotinate derivatives and by their gas-liquid chromatographic retention times. In meibomian gland lipids from the mouse, cholesterol was the major constituent; alcohols from the n-, iso-, anteiso- and unsaturated series were present with iso-C26 and anteiso-C27 being the most abundant. Mono-unsaturated fatty acids belonged mainly to the omega 9 series and saturated acids belonged to the iso-, anteiso- and n-series. Several 1,2-diols were also identified. The most abundant of these had iso-C16 and iso-C20 chains. GC-MS studies on the intact wax esters showed them to be composed of the branched-chain alcohols and both branched-chain and unsaturated acids.

Double-bond patterns of fatty acids and alcohols in steer and human meibomian gland lipids.

Ozonolysis studies of the monoenes of the fatty chain types in lipids of steer meibomian gland excreta (meibum) have confirmed earlier structural assignments based on gas liquid chromatography (GLC) retention data and have assisted in assigning complete structures to a group of recently identified omega-hydroxy fatty acids. The omega-hydroxy acids include straight-chain monoenoic acids (85%), saturated anteiso and iso acids (13%), monoenoic acids of the latter group (1%) and, finally, saturates of the normal monoenoic acids (1%). All the fatty chains of meibum can be biosynthesized by a unified process of chain buildup to primary chain lengths of 12:0-20:0 for the straight evens, with 16:0 predominating, 13:0-21:0 for the straight odds with 17:0 predominating, i16:0 to i28:0 for the iso and ai17:0 to ai29:0 for the anteiso chain types; then delta 9 desaturation of each of these chain types: and finally chain elongation of 1-10 C2 units. Some chain degradation may also occur. The meibum lipid components involved are unsubstituted fatty acids, alpha-OH fatty acids, alpha-OH fatty acids, omega-OH fatty acids, fatty alcohols and some other lipid components incompletely characterized. The carbon skeletons are straight even, straight odd, iso and anteiso except that the alpha-OH fatty acids are only straight even and straight odd and these chains are not elongated. All fatty chains are almost entirely saturated and monoenoic, the polyenes occurring in only trace amounts. Biosynthesis of the fatty chains of human meibum evidently occurs similarly, except that considerably more 18:0 than 16:0 fatty acids are built up by the fatty acid synthetase, before desaturation and extension.

[Immunofluorescence study of various constituents of the eyelids in rats before, during and after their closure]. / Etude par immunofluorescence de différents constituants des paupières de rat avant, pendant et après leur ferméture.

We have brought the evidence for the presence of type I and III collagens and laminin in cultures of eyelid buds removed just before the onset of their junction. The patterns of distribution of laminin (localized in all basement membranes) and of the type III collagen remain constant during all the culture. Type I collagen is distributed homogeneously in the mesenchyma like type III collagen after the twelfth hour of culture. The rapid stabilization of these three distributions would be associated with the persistence of an epithelial seam limited by intact basement membranes between mesenchymal eyelids from the stage of junction to the later stage of disjunction of the eyelids.

Meibomian gland studies: comparison of steer and human lipids.

Simple surgery gave whole meibomian glands of steer yielding approximately 47 mg of lipid per pair of lids. This lipid gave a thin-layer chromatography pattern similar to that of steer or human excreta. Column chromatography of pooled steer gland lipids and pooled human lipid (excreta) gave 0.06% and 7.54% hydrocarbons, respectively, containing no squalene. Excluding human hydrocarbons (as exogenous material) the percentage composition of steer and human lipids were, respectively, sterol esters 31.7 and 29.5; wax esters 31.2 and 35.0; material in the diester region 11.4 and 8.4; triacyl glycerols 1.6 and 4.0; material in the post-triacyl glycerol region 2.8 and 3.2; free sterols 3.0 and 1.8; free fatty acids 5.1 and 2.1; and polar lipids 13.3 and 16.0. Glass capillary gas-liquid chromatography revealed the following: a series of normal odd- and even-chain hydrocarbons (C16 to C32) for the steer and a complex pattern for the human samples; unsubstituted fatty acids in total lipids and various lipid classes ranging from C12 to C31 with n-even, n-odd, iso-even and iso-odd, and anteiso-odd chains for both samples; and fatty alcohols in total lipids and wax esters ranging from C18 to C31. Anteiso structures predominated in the steer, and iso structures in the human samples. Steer fatty acids favored anteiso branching and saturation, whereas human acids favored iso branching and unsaturation. Fatty acids of sterol esters in both samples showed a wider range of chain lengths than did other lipid classes. Similarity of the fatty acid pattern suggests that the free fatty acids are derived from the triacyl glycerols as in human sebum. Identification of saturated fatty acids was confirmed by a gas liquid chromatography--mass spectrometry--data system (GLC-MS-DS). We define "meibum" and "meibocyte" as the meibomian gland equivalent of sebum and sebocyte.

Natural fat in external eye. Vital-stained by Sudan III powder.

Examination of 113 eyes vital-stained by Sudan powder revealed that fat on the skin of outer canthus may pass onto the precorneal film (in 68%), and that the most reliable staining is obtained by application on the bulbar conjunctiva below the limbus corneae at 6 o'clock. In 41%, the precorneal film had stained drops or foam, with a tendency towards an increase among pathological cases (infectious conjunctivitis and blepharitis). An increased amount of fat was found in the mucous thread of eyes affected with infectious conjunctivitis and lagophthalmos. In a small number of pathological cases fat was also detected on cornea, bulbar conjunctiva, and inferior fornix. The fatty layer on the lower lid margin was stained more frequently and more intensely than that on the superior. The secretion of the meibomian glands was stained in no more than a scant one half of the glands (on an average 44%), independently of age, sex, site, and diagnosis.

Application of combined liquid chromatography/mass spectrometry (LC/MS): analysis of petroporphyrins and meibomian gland waxes.

Application of the moving belt LC/MS interface is illustrated by two diverse examples, one from the field of petroporphyrin chemistry, the other, a sample of high molecular weight waxes from the steer meibomian gland (eye). Petroporphyrins are difficult of convert to derivatives suitable for GC analysis and the advantages of analysis by a combined separation/identification method are well demonstrated by the LC/MS runs. High molecular weight waxes can be saponified and derivatized for other analyses such as GC and GC/MS, but information regarding the distribution of alcohol and acid in the combined state is destroyed. This study shows that such information can be obtained by a combined LC/MS analysis without chemical manipulation of the sample.

Composition of the neutral lipids of bovine meilbomian secretions.

Lipids secreted form the bovine meibomian glands were assigned to following classes: cholesteryl esters (A) (fatty acyl chain lengths from 15 to 27 carbon atoms), 41%; wax esters, 29%; triacylglycerols, 10%; and cholesteryl ester (B) (fatty acyl chain lengths from 14 to 18 carbons), 15%. The remaining 5% consisted of cholesterol, fatty acids, and highly polar material. Analysis of the lipids showed that only the cholesteryl esters (A) contained fatty acyl chains greater than or equal to C20:0. One third of the fatty acyl chains of the wax esters and the cholesteryl esters (B) was iC15:0. The fatty alcohol moieties of the wax esters were found to be branched chain, C23:0-C27:0. A computer-based programmable gas-liquid chromatographic procedure using 10% apolar 10C on Gas Chrom Q could be used to distinguish both iso and anteiso fatty alcohols and esters of fatty acids.