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. 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. Due to a lack of turnover (1Lynnerup N. Kjeldsen H. Heegaard S. Jacobsen C. Heinemeier J. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life.PLoS ONE. 2008; 3: e1529Crossref PubMed Scopus (174) Google Scholar), the lens is an ideal tissue for examining age-related changes to biomolecules, and characterizing these can have clinical relevance for understanding the molecular basis of ocular conditions such as presbyopia (2Glasser A. Campbell M.C.W. Biometric, optical and physical changes in the isolated human crystalline lens with age in relation to presbyopia.Vision Res. 1999; 39: 1991-2015Crossref PubMed Scopus (323) Google Scholar) and nuclear cataract (3Spector A. Oxidation and cataract.Ciba Found. Symp. 1984; 106: 48-64PubMed Google Scholar, 4Truscott R.J.W. Age-related nuclear cataract - oxidation is the key.Exp. Eye Res. 2005; 80: 709-725Crossref PubMed Scopus (663) Google Scholar). It is not known which are the critical factors predisposing the human lens to age-related nuclear (ARN) cataract. Research from one of us, over many years, suggests that the formation of an internal barrier to the diffusion of small molecules may be a key event in the onset of this pathology (5Moffat B.A. Landman K.A. Truscott R.J.W. Sweeney M.H.J. Pope J.M. Age-related changes in the kinetics of water transport in normal human lenses.Exp. Eye Res. 1999; 69: 663-669Crossref PubMed Scopus (96) Google Scholar, 6Sweeney M.H.J. Truscott R.J.W. An impediment to glutathione diffusion in older normal human lenses: a possible precondition for nuclear cataract.Exp. Eye Res. 1998; 67: 587-595Crossref PubMed Scopus (185) Google Scholar). The barrier forms in the normal lens at middle age and uncouples the center of the lens from the metabolically active outer region. Because the lens grows continuously throughout life, the outmost part of the lens is the region that was formed most recently and has the highest concentration of active enzymes (7Dovrat A. Scharf J. Gershon D. Glyceraldehyde-3-phosphate dehydrogenase-activity in rat and human lenses and the fate of enzyme molecules in the aging lens.Mech. Ageing Dev. 1984; 28: 187-191Crossref PubMed Scopus (31) Google Scholar, 8Zhang W.Z. Augusteyn R.C. Aging of glutathione-reductase in the lens.Exp. Eye Res. 1994; 59: 91-95Crossref PubMed Scopus (30) Google Scholar). It is where the major antioxidant glutathione (GSH) is synthesized and the oxidized form of glutathione rereduced (9Giblin F.J. Glutathione: a vital lens antioxidant.J. Ocul. Pharmacol. Ther. 2000; 16: 121-135Crossref PubMed Scopus (322) Google Scholar, 10Rathbun W.B. Bovis M.G. Holleschau A.M. Species survey of glutathione peroxidase and glutathione reductase: search for an animal model of the human lens.Ophthalmic Res. 1986; 18: 282-287Crossref PubMed Scopus (23) Google Scholar). Since GSH is essential for maintaining a reducing environment in the lens center and for protection of protein structure, formation of the barrier allows oxidative modification of nuclear proteins that is the hallmark of nuclear cataract. The dimensions of the barrier region (7 mm equatorial × 3 mm axial) corresponds to the part of the adult lens that was synthesized immediately after birth (11Kuszak J.R. The development of lens sutures.Prog. Retin. Eye Res. 1995; 14: 567-592Crossref Scopus (46) Google Scholar, 12Garland D.L. Duglas-Tabor Y. Jimenez-Asensio J. Datiles M.B. Magno B. Nucleus of the human lens: demonstration of a highly characteristic protein pattern by two-dimensional electrophoresis and a new method of lens dissection.Exp. Eye Res. 1996; 62: 285-291Crossref PubMed Scopus (61) Google Scholar). Recent data implicate the binding of denatured proteins to the fiber cell membrane as the mechanism responsible for the barrier (13Friedrich M.G. Truscott R.J.W. Membrane association of proteins in the aging human lens: profound changes take place in the fifth decade of life.Invest. Ophthalmol. Vis. Sci. 2009; 50: 4786-4793Crossref PubMed Scopus (43) Google Scholar), possibly by occluding the membrane pores that normally facilitate the movement of GSH and water from cell to cell via connexon (14Jacobs M.D. Soeller C. Sisley A.M. Cannell M.B. Donaldson P.J. Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.Invest. Ophthalmol. Vis. Sci. 2004; 45: 191-199Crossref PubMed Scopus (49) Google Scholar) and aquaporin 0 (15Gonen T. Cheng Y. Sliz P. Hiroaki Y. Fujiyoshi Y. Harrison S.C. Walz T. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.Nature. 2005; 438: 633-638Crossref PubMed Scopus (532) Google Scholar) channels, respectively. Moreover, aquaporin 0 requires interaction with distinct membrane lipids to form its correct functional conformation (15Gonen T. Cheng Y. Sliz P. Hiroaki Y. Fujiyoshi Y. Harrison S.C. Walz T. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.Nature. 2005; 438: 633-638Crossref PubMed Scopus (532) Google Scholar). If interactions with fiber cell membranes are involved in barrier formation, it is clearly important to characterize age-related changes to membrane composition. This study employed matrix-assisted laser desorption ionization (MALDI) imaging and electrospray-ionization mass spectrometry (ESI-MS) to examine the phospholipid composition within defined regions of the human lens. Human lenses (ages 23–70, male) were obtained from the Sydney Lions Eye Bank, Sydney, NSW, Australia. All work was approved by the human research ethics committees at the University of Sydney (#7292) and the University of Wollongong (HE 99/001). Lenses were sliced using a cryostat (Leica, 1720) at −20°C. Sufficient TissueTekTM was applied to adhere the lens to the cutting block in the correct orientation (i.e., the lens was not completely submerged in TissueTekTM). Transverse slices of the lenses (10–25 μm thick) were placed directly onto glass cover slips and stored at −20°C until analyzed. Imaging of human lens lipids was executed as previously described for other tissues (16Hankin J.A. Barkley R.M. Murphy R.C. Sublimation as a method of matrix application for mass spectrometric imaging.J. Am. Soc. Mass Spectrom. 2007; 18: 1646-1652Crossref PubMed Scopus (444) Google Scholar). In brief, double-sided, heat-conducting tape was used to adhere the underside of each glass cover slip to a steel plate. The steel plate was inverted and placed on the cold finger of a glass sublimation device with the lens slice exposed, and dihydroxybenzoic acid (275 mg) was sublimed for 11 min. Following sublimation of the matrix, the heat-conducting tape was removed, and the lens slices were analyzed using a QSTAR XLTM (Applied Biosystems/MDS Sciex, Thornhill, Ontario, Canada) quadrupole-TOF mass spectrometer fitted with an orthogonal MALDI source. Positive ion images m/z 350–1000 were obtained using a spatial resolution of 50 μm. Mass spectra were acquired using 10 laser pulses per image spot with a 0.5 s accumulation time. Collision-induced dissociation was used for structural confirmation of abundant lipids observed with sphingomyelin ions identified by the observation of the characteristic phosphocholine fragment ion at m/z 184. Data were analyzed using Applied Biosystems Analyst QSTM and BiomapsTM version 3.7.5.5 software (Novartis, Basel, Switzerland). Averaged MALDI spectra for the outer, barrier, and nuclear regions of the lens were obtained by selecting the region of interest (ROI) according to the dimensions shown in Fig. 1B within the BiomapsTM software. The counts of the [DHSM (d18:0/ 16:0) + H]+ (m/z 705) and [DHCer (d18:0/16:0) + H]+ (m/z 540) ions obtained from these regionally averaged spectra were used to create graphs (see Fig. 6A, C, of the ion of [DHSM (d18:0/ 16:0) + H]+ obtained from MALDI analysis of lens slices from 23-year-old and 70-year-old with an analysis of lenses from and A of the ion of [DHCer (d18:0/16:0) + H]+ obtained from MALDI analysis of lens slices from 23-year-old and 70-year-old with an analysis of lenses from and is as an ion is as a of the internal for that lipid image of human lenses and years were into regions as shown in Fig. In brief, human lenses were using cold into barrier, and outer regions of and respectively. The lens regions from each were and the were as described from the lens regions were by the method of S. H. Y. quantitative for analysis of by mass 2007; PubMed Scopus Google Scholar) with In brief, lenses were and of was to glass each lens A each of the internal and DHSM was to the tissues at per of lens The were and at was to each to at for the of acid was by of and of The was and at for min. The was and and the was to at The was in of and stored at until was with and was at 50 were into the ion at a of 10 using the and mass spectra acquired as previously described J.M. J.R. J. Truscott R.J.W. Human lens lipids from of used 2008; PubMed Scopus Google Scholar). All mass spectra were obtained using a with a ion and by version software. was to to and to was to and in and ion respectively. was used as the at a of and as the at a of 3 were identified by ion in ion for the phosphocholine (m/z at and the (m/z at 50 DHSM (d18:0/16:0) ion are as a of the DHSM internal using m/z ion and were by for and in ion using a of DHCer (d18:0/16:0) ion are as a of the internal using ion mass All ion were after for spectrometry analysis was on the extracts (see for confirmation of the and These were on an mass spectrometer (Applied Biosystems/MDS Sciex, Thornhill, Ontario, Canada) in ion m/z m/z and m/z within one The conditions were ion and conditions were applied to from sphingomyelin molecules as described by S. H. Y. quantitative for analysis of by mass 2007; PubMed Scopus Google Scholar). conditions involved a × 3 μm of with of with at for to in 10 to in min. Human lenses the ages of and years were into regions as described in with M.G. Truscott R.J.W. and water in normal and human Ophthalmol. Vis. Sci. 2008; PubMed Scopus (46) Google Scholar). The barrier regions were and on as part of a study to the effect of age on the membrane binding of proteins (13Friedrich M.G. Truscott R.J.W. Membrane association of proteins in the aging human lens: profound changes take place in the fifth decade of life.Invest. Ophthalmol. Vis. Sci. 2009; 50: 4786-4793Crossref PubMed Scopus (43) Google Scholar). lipids from each were by the method of J. A method for the and of from animal PubMed Google Scholar) with J.M. J.R. J. Truscott R.J.W. Human lens lipids from of used 2008; PubMed Scopus Google Scholar). and of in these extracts was using analysis as described from have shown that the DHSM (d18:0/16:0) is the most abundant phospholipid in the human of J.M. J.R. J. Truscott R.J.W. Human lens lipids from of used 2008; PubMed Scopus Google Scholar, D. of sphingomyelin and of human lens Res. PubMed Scopus Google Scholar, A. D. of the phospholipid composition in membranes of adult human lenses by and MALDI PubMed Scopus Google Scholar). Fig. a ion MALDI mass acquired from tissue sliced from a 70-year-old human lens. The most abundant ions are of (m/z and the (m/z (m/z and (m/z ions of The of the ion at m/z was by of a dissociation mass that revealed the phosphocholine (m/z as the major ion The in of the m/z ion tissue slices from one years and and years human lenses is in Fig. were observed for the and ions and that these images are of the distribution of the lipid within the The data in Fig. 3 that the distribution of DHSM (d18:0/16:0) is in the 23-year-old is a distinct annular distribution of this lipid observed in older lenses. MALDI analysis of lens tissue also revealed a ion at m/z to the ion of the dihydrosphingomyelin, DHSM the ion was to the directly by the distribution observed for this ion were to of m/z in and lens tissue sections Fig. ion MALDI images the distribution of the ion at m/z to [DHSM (d18:0/16:0) + H]+ in slices from and 70-year-old human lenses. The regions of highest ion are shown in the to is (see key The tissue slices in these were image The of mass spectrometer used for MALDI imaging in this study a mass for each on the tissue that is by the the used to the images in Fig. 3 be for ions that were to of m/z These revealed an ion at m/z of to m/z was in the older lenses to have a distribution to that of DHSM The MALDI images of m/z in the lens sections are shown in Fig. and reveal that this ion is in the 23-year-old in the 64- and 70-year-old it is most abundant in the nuclear region in the outer regions of the lens. m/z corresponds in mass to the ion of the dihydroceramide, DHCer (d18:0/16:0), the structural of the most abundant lens phospholipid DHSM the of this ion not be directly from the MALDI analysis of lipid extracts of tissue from the nucleus of other lenses revealed the ions at m/z that were also in for which are for and of molecular from lipid extracts of by ionization mass PubMed Scopus Google Scholar). These ion also identified the of the DHCer with an ion at m/z The ion at m/z was in in the MALDI analysis but revealed a distribution to that of DHCer (d18:0/16:0) and to the DHSM Fig. The of DHCer (d18:0/16:0) and DHCer in lens tissue was by analysis of extracts from nuclear tissue using a normal as described by S. H. Y. quantitative for analysis of by mass 2007; PubMed Scopus Google Scholar). these were from the and and were clearly from that and min. The DHCer (d18:0/16:0) and DHCer were clearly identified in the analysis by ion for m/z that ions at m/z and Fig. analysis also revealed the of ions at m/z and from the forms of these The m/z ion is also in the MALDI shown in Fig. and of its distribution not revealed a pattern to that of m/z for the of the ion as [DHCer (d18:0/16:0) + H]+ within the MALDI ion ESI mass spectra of the lipid from a lens region. A mass and ions that a of dissociation This a that of the DHCer in the lipid image the images of DHSM (d18:0/ 16:0) and DHCer (d18:0/16:0) obtained from the human lens tissue sections were of the distribution of the lipids in an analysis of these tissues was Lenses obtained from and were dissected as described in and the of DHSM (d18:0/16:0) and DHCer (d18:0/16:0) were in each region by of the tissue and analysis in the of internal Fig. Fig. a of the obtained from the MALDI mass spectra obtained over the regions of the lens with the of the lipids obtained by of dissected lenses. The [DHSM (d18:0/16:0) + H]+ ion counts obtained from each region of the MALDI image data are in Fig. years and Fig. years The ion distribution of the regions obtained from MALDI imaging a to the data obtained from the study for the and lenses. In the lenses a distribution of DHSM (d18:0/16:0) throughout the the older lenses of of this lipid in the barrier region. This that the distribution of the m/z ion as observed by MALDI imaging is of the distribution of the DHSM (d18:0/16:0) lipid within the lens and is not an from changes in lens in the of the MALDI The the MALDI imaging data and that obtained from the lipid extracts also for DHCer (d18:0/16:0), as can be in DHCer (d18:0/16:0) was in the lenses the on the older lenses with DHCer (d18:0/16:0) in the shown in Fig. to an increase in DHSM (d18:0/16:0) in the barrier region with is data of and to age-related in molecular composition of the lens. In the lack of lenses from in the of this study was to be extracts from the barrier region of the lenses of 18 in age from to years, were obtained from a study the membrane association of lens proteins (13Friedrich M.G. Truscott R.J.W. Membrane association of proteins in the aging human lens: profound changes take place in the fifth decade of life.Invest. Ophthalmol. Vis. Sci. 2009; 50: 4786-4793Crossref PubMed Scopus (43) Google Scholar). and analysis of these was in the study, and the of sphingomyelin was to internal In with the data described this an increase in the sphingomyelin in the barrier region and 40 years of age. This is by in sphingomyelin concentration the ages of 40 and MALDI mass spectrometric imaging of lens sections revealed a in the distribution of lipids in older human lenses. In a of DHSM concentration was observed in lenses older years, and its dimensions in to that of the barrier to diffusion (5Moffat B.A. Landman K.A. Truscott R.J.W. Sweeney M.H.J. Pope J.M. Age-related changes in the kinetics of water transport in normal human lenses.Exp. Eye Res. 1999; 69: 663-669Crossref PubMed Scopus (96) Google Scholar, 6Sweeney M.H.J. Truscott R.J.W. An impediment to glutathione diffusion in older normal human lenses: a possible precondition for nuclear cataract.Exp. Eye Res. 1998; 67: 587-595Crossref PubMed Scopus (185) Google Scholar). The of in this were using quantitative on ionization mass The region of DHSM concentration was not in lenses. obtained MALDI images of lipids within the lens of in the nucleus the but distinct is observed in the lipid distribution J. P. J.M. spatial lipid distribution in lens by MALDI imaging mass Res. of PubMed Scopus (46) Google Scholar) to that The barrier to which in normal human lenses at middle has R.J.W. Age-related nuclear cataract - oxidation is the key.Exp. Eye Res. 2005; 80: 709-725Crossref PubMed Scopus (663) Google Scholar) to be the for the onset of the cataract. the of small molecular such as the antioxidant from its place of in the lens into the lens the proteins in the center of the lens to oxidative which is the hallmark of the cataract (3Spector A. Oxidation and cataract.Ciba Found. Symp. 1984; 106: 48-64PubMed Google Scholar, R.J.W. Augusteyn R.C. changes in human lens proteins during nuclear cataract PubMed Scopus Google Scholar). are possible for the age-related increase in sphingomyelin in the barrier region observed in this The is that the fiber cell membranes of the barrier region a with the sphingomyelin at the of other membrane have identified an increase in the sphingomyelin to in lenses Y. D. D. Human lens phospholipid changes with age and Ophthalmol. Vis. Sci. 2005; PubMed Scopus Google Scholar, P. T. Human crystalline lens phospholipid analysis with Ophthalmol. Vis. Sci. Google Scholar) and also in the lens and nucleus D. with human and lens Eye Res. PubMed Scopus Google Scholar). these and other changes in the ocular lens have the of a D. and the ocular Res. of PubMed Scopus Google Scholar). data the age and in the barrier region. that age-related in concentration are also observed in regions of the rat with increase in a in of aging on the composition and of sphingomyelin in the PubMed Scopus Google Scholar). An for the age-related increase in sphingomyelin in the that lipid levels in this study were as a of tissue the of internal was by tissue mass (see a in the mass of the barrier region also in an observed increase in The to tissue mass in the lens are protein and water in the nuclear to throughout M.G. Truscott R.J.W. and water in normal and human Ophthalmol. Vis. Sci. 2008; PubMed Scopus (46) Google Scholar), is a of in protein concentration the ages of and This is not to the of sphingomyelin levels observed in the barrier region ages and 40 The part of the human which is approximately mm in equatorial corresponds to the of the lens at birth D.L. Duglas-Tabor Y. Jimenez-Asensio J. Datiles M.B. Magno B. Nucleus of the human lens: demonstration of a highly characteristic protein pattern by two-dimensional electrophoresis and a new method of lens dissection.Exp. Eye Res. 1996; 62: 285-291Crossref PubMed Scopus (61) Google Scholar) and is not during adult M.G. Truscott R.J.W. and water in normal and human Ophthalmol. Vis. Sci. 2008; PubMed Scopus (46) Google Scholar, B. human lens - the distribution of Eye Res. PubMed Scopus Google Scholar, The human lens. on the lens.Exp. Eye Res. 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This was most in the nuclear region. the and composition of the to of human lens the most for the of the is that are by of the The data also that this is associated with age. In the region of the is protein turnover (1Lynnerup N. Kjeldsen H. Heegaard S. Jacobsen C. Heinemeier J. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life.PLoS ONE. 2008; 3: e1529Crossref PubMed Scopus (174) Google it is that are involved this that enzyme molecules active for this it is that of in older lenses an of DHSM in this environment over a This is it is not known the are to within the the known of these molecules that in older lenses may be involved in other at in cell are to cell of lens A. cell in lens Vis. 2007; Google Scholar) and have to a in the formation of cataract. work has changes in the lipid composition of the human lens with age. The of fiber in to the of a of DHSM into may one for the of binding of normally lens onto the fiber cell membranes in lenses (13Friedrich M.G. Truscott R.J.W. Membrane association of proteins in the aging human lens: profound changes take place in the fifth decade of life.Invest. Ophthalmol. Vis. Sci. 2009; 50: 4786-4793Crossref PubMed Scopus (43) Google Scholar). The are to for in of of the with age-related nuclear cataract

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