Instability of the cellular lipidome with age.

The human lens nucleus is formed in utero, and from birth onwards, there appears to be no significant turnover of intracellular proteins or membrane components. Since, in adults, this region also lacks active enzymes, it offers the opportunity to examine the intrinsic stability of macromolecules under physiological conditions. Fifty seven human lenses, ranging in age from 12 to 82 years, were dissected into nucleus and cortex, and the nuclear lipids analyzed by electrospray ionization tandem mass spectrometry. In the first four decades of life, glycerophospholipids (with the exception of lysophosphatidylethanolamines) declined rapidly, such that by age 40, their content became negligible. In contrast the level of ceramides and dihydroceramides, which were undetectable prior to age 30, increased approximately 100-fold. The concentration of sphingomyelins and dihydrosphingomyelins remained unchanged over the whole life span. As a consequence of this marked alteration in composition, the properties of fiber cell membranes in the centre of young lenses are likely to be very different from those in older lenses. Interestingly, the identification of age 40 years as a time of transition in the lipid composition of the nucleus coincides with previously reported macroscopic changes in lens properties (e.g., a massive age-related increase in lens stiffness) and related pathologies such as presbyopia. The underlying reasons for the dramatic change in the lipid profile of the human lens with age are not known, but are most likely linked to the stability of some membrane lipids in a physiological environment.

Imaging of human lens lipids by desorption electrospray ionization mass spectrometry.

The lipid composition of the human lens is distinct from most other tissues in that it is high in dihydrosphingomyelin and the most abundant glycerophospholipids in the lens are unusual 1-O-alkyl-ether linked phosphatidylethanolamines and phosphatidylserines. In this study, desorption electrospray ionization (DESI) mass spectrometry-imaging was used to determine the distribution of these lipids in the human lens along with other lipids including, ceramides, ceramide-1-phosphates, and lyso 1-O-alkyl ethers. To achieve this, 25 µm lens slices were mounted onto glass slides and analyzed using a linear ion-trap mass spectrometer equipped with a custom-built, 2-D automated DESI source. In contrast to other tissues that have been previously analyzed by DESI, the presence of a strong acid in the spray solvent was required to desorb lipids directly from lens tissue. Distinctive distributions were observed for [M + H](+) ions arising from each lipid class. Of particular interest were ionized 1-O-alkyl phosphatidylethanolamines and phosphatidylserines, PE (18:1e/18:1), and PS (18:1e/18:1), which were found in a thin ring in the outermost region of the lens. This distribution was confirmed by quantitative analysis of lenses that were sectioned into four distinct regions (outer, barrier, inner, and core), extracted and analyzed by electrospray ionization tandem mass spectrometry. DESI-imaging also revealed a complementary distribution for the structurally-related lyso 1-O-alkyl phosphatidylethanolamine, LPE (18:1e), which was localized closer to the centre of the lens. The data obtained in this study indicate that DESI-imaging is a powerful tool for determining the spatial distribution of human lens lipids.

Sphingolipid distribution changes with age in the human lens.

The formation of an internal barrier to the diffusion of small molecules in the lens during middle age is hypothesized to be a key event in the development of age-related nuclear (ARN) cataract. Changes in membrane lipids with age may be responsible. In this study, we investigated the effect of age on the distribution of sphingomyelins, the most abundant lens phospholipids. Human lens sections were initially analyzed by MALDI mass spectrometry imaging. A distinct annular distribution of the dihydrosphingomyelin, DHSM (d18:0/16:0), in the barrier region was observed in 64- and 70-year-old lenses but not in a 23-year-old lens. An increase in the dihydroceramide, DHCer (d18:0/16:0), in the lens nucleus was also observed in the older lenses. These findings were supported by ESI mass spectrometry analysis of lipid extracts from lenses dissected into outer, barrier, and nuclear regions. A subsequent analysis of 18 lenses ages 20-72 years revealed that sphingomyelin levels increased with age in the barrier region until reaching a plateau at approximately 40 years of age. Such changes in lipid composition will have a significant impact on the physical properties of the fiber cell membranes and may be associated with the formation of a barrier.

Identification of abundant alkyl ether glycerophospholipids in the human lens by tandem mass spectrometry techniques.

Previous studies have shown that the human lens contains glycerophospholipids with ether linkages. These lipids differ from conventional glycerophospholipids in that the sn-1 substituent is attached to the glycerol backbone via an 1-O-alkyl or an 1-O-alk-1'-enyl ether rather than an ester bond. The present investigation employed a combination of collision-induced dissociation (CID) and ozone-induced dissociation (OzID) to unambiguously distinguish such 1-O-alkyl and 1-O-alk-1'-enyl ethers. Using these methodologies the human lens was found to contain several abundant 1-O-alkyl glycerophosphoethanolamines, including GPEtn(16:0e/9Z-18:1), GPEtn(11Z-18:1e/9Z-18:1), and GPEtn(18:0e/9Z-18:1), as well as a related series of unusual 1-O-alkyl glycerophosphoserines, including GPSer(16:0e/9Z-18:1), GPSer(11Z-18:1e/9Z-18:1), GPSer(18:0e/9Z-18:1) that to our knowledge have not previously been observed in human tissue. Isomeric 1-O-alk-1'-enyl ethers were absent or in low abundance. Examination of the double bond position within the phospholipids using OzID revealed that several positional isomers were present, including sites of unsaturation at the n-9, n-7, and even n-5 positions. Tandem CID/OzID experiments revealed a preference for double bonds in the n-7 position of 1-O-ether linked chains, while n-9 double bonds predominated in the ester-linked fatty acids [e.g., GPEtn(11Z-18:1e/9Z-18:1) and GPSer(11Z-18:1e/9Z-18:1)]. Different combinations of these double bond positional isomers within chains at the sn-1 and sn-2 positions point to a remarkable molecular diversity of ether-lipids within the human lens.

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.

Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions.

Ions formed from lipids during electrospray ionization of crude lipid extracts have been mass-selected within a quadrupole linear ion trap mass spectrometer and allowed to react with ozone vapor. Gas-phase ion-molecule reactions between unsaturated lipid ions and ozone are found to yield two primary product ions for each carbon-carbon double bond within the molecule. The mass-to-charge ratios of these chemically induced fragments are diagnostic of the position of unsaturation within the precursor ion. This novel analytical technique, dubbed ozone-induced dissociation (OzID), can be applied both in series and in parallel with conventional collision-induced dissociation (CID) to provide near-complete structural assignment of unknown lipids within complex mixtures without prior fractionation or derivatization. In this study, OzID is applied to a suite of complex lipid extracts from sources including human lens, bovine kidney, and commercial olive oil, thus demonstrating the technique to be applicable to a broad range of lipid classes including both neutral and acidic glycerophospholipids, sphingomyelins, and triacylglycerols. Gas-phase ozonolysis reactions are also observed with different types of precursor ions including [M+H]+, [M+Li]+, [M+Na]+, and [M-H]-: in each case yielding fragmentation data that allow double bond position to be unambiguously assigned. Within the human lens lipid extract, three sphingomyelin regioisomers, namely SM(d18:0/15Z-24:1), SM(d18:0/17Z-24:1), and SM(d18:0/19Z-24:1), and a novel phosphatidylethanolamine alkyl ether, GPEtn(11Z-18:1e/9Z-18:1), are identified using a combination of CID and OzID. These discoveries demonstrate that lipid identification based on CID alone belies the natural structural diversity in lipid biochemistry and illustrate the potential of OzID as a complementary approach within automated, high-throughput lipid analysis protocols.

Elucidation of double bond position in unsaturated lipids by ozone electrospray ionization mass spectrometry.

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication [Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduct ion (e.g., [M - H](-), [M - 2H](2-), [M + H](+), [M + Na](+), [M + NH(4)](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.