Changes in the Properties and Organization of Human Lens Lipid Membranes Occurring with Age

Abstract

Purpose: This research was undertaken to document the changes in the organization and properties of human lens lipid membranes that occur with age.

Methods: Human lens lipid membranes prepared from the total lipids extracted from clear lens cortices and nuclei of donors from age groups 0-20 and 21-40 years were investigated. An electron paramagnetic resonance technique and nitroxide spin labels (analogues of phospholipids and cholesterol) were used.

Results: Two distinct lipid domains, the phospholipid-cholesterol domain (PCD) and the pure cholesterol bilayer domain (CBD), were detected in all investigated membranes. Profiles of the acyl chain order, fluidity, hydrophobicity, and oxygen transport parameter across discriminated coexisting lipid domains were assessed. Independent of the age-related changes in phospholipid composition, the physical properties of the PCD remained the same for all age groups and were practically identical for cortical and nuclear membranes. However, the properties of pure CBDs changed significantly with the age of the donor and were related to the size of the CBD, which increased with the age of the donor and was greater in nuclear than in cortical membranes. A more detailed analysis revealed that the size of the CBD was determined mainly by the cholesterol content in the membrane.

Conclusions: This paper presents data from four age groups: 0-20, 21-40, 41-60, and 61-70 years. Data from age groups 41-60 and 61-70 years were published previously. Combining the previously published data with those data obtained in the present work allowed us to show the changes in the organization of cortical and nuclear lens lipid membranes as functions of age and cholesterol. It seems that the balance between age-related changes in membrane phospholipid composition and cholesterol content plays an integral role in the regulation of cholesterol-dependent processes in fiber cell membranes and in the maintenance of fiber cell membrane homeostasis.

Keywords: Cholesterol; cholesterol bilayer domain; cholesterol crystals; membrane domains; spin labeling.

Figure 1

Chemical structures of phospholipid- (n-PCs, T-PC, 9-SASL) and Chol-analogue spin labels (CSL, ASL), together with the structure of phosphatidylcholine (POPC), palmitoyl sphingomyelin (SM) and palmitoyl dihydrosphingomyelin (DHSM) (SM and DHSM are the most abundant phospholipids in human lens membranes), and cholesterol (CHOL). The approximate locations of these molecules across the lipid bilayer membrane are illustrated.

Figure 2

Profiles of the order parameter obtained at 37°C with phospholipid-analogue spin labels across PCD for cortical (A) and nuclear (B) lens lipid membranes of eye lenses from human donors of different age groups. Data for age groups 41–60 and 61–70 years are reproduced, respectively, from Reference , Copyright 2013, with permission from Elsevier, and from Reference , Copyright 2014, with permission from Springer Publishing. The approximate localizations of the nitroxide moieties of spin labels are indicated by arrows.

Figure 3

Fluidity profiles (T₁⁻¹ versus depth in the membrane) obtained at 37°C with phospholipid-analogue spin labels across PCD for cortical (A) and nuclear (B) lens lipid membranes of eye lenses from human donors of different age groups. Data for age groups 41–60 and 61–70 years are reproduced, respectively, from Reference , Copyright 2013, with permission from Elsevier, and from Reference , Copyright 2014, with permission from Springer Publishing. Values in the headgroup region were obtained with the T-PC (T), the nitroxide moiety of which possesses a different chemical structure than nitroxide moieties of other phospholipid-analogue spin labels (Fig. 1). Because of that, values obtained with T-PC cannot be straightforward compared with those obtained in the membrane hydrocarbon phase (see Sect. 3.1.5). The approximate localizations of the nitroxide moieties of spin labels are indicated by arrows.

Figure 4

Profiles of the OTP (the local oxygen diffusion-concentration product) obtained at 37°C with phospholipid- (solid symbols) and Chol-analogue spin labels (open symbols) across PCD (solid lines) and CBD (broken lines) for cortical (A) and nuclear (B) lens lipid membranes of eye lenses from human donors of different age groups. Data for age groups 41–60 and 61–70 years are reproduced, respectively, from Reference , Copyright 2013, with permission from Elsevier, and from Reference , Copyright 2014, with permission from Springer Publishing. The approximate localizations of the nitroxide moieties of spin labels are indicated by arrows. Localizations of the nitroxide moieties of Chol analogue spin labels of ASL and CSL in the PCD and the CBD are explained in Sect. 3.5 of the reference. The value of the OTP in the aqueous phase also is shown (dotted lines).

Figure 5

Hydrophobicity profiles (2A_Z versus depth in the membrane) obtained with phospholipid-analogue spin labels across PCD for cortical (A) and nuclear (B) lens lipid membranes of eye lenses from human donors of different age groups. Data for age groups 41–60 and 61–70 years are reproduced, respectively, from Reference , Copyright 2013, with permission from Elsevier, and from Reference , Copyright 2014, with permission from Springer Publishing. Values in the headgroup region were obtained with the T-PC (T), the nitroxide moiety of which possesses a different chemical structure than the nitroxide moieties of other phospholipid-analogue spin labels (Fig. 1). Because of that, values obtained with T-PC cannot be straightforward compared with those obtained in the membrane hydrocarbon phase. The 2A_Z value in the aqueous phase (a dotted line) was obtained with 16-SASL; thus, it can be straightforward compared with those in the hydrocarbon phase. (See Sect. 3.1.5 for more explanation.) Approximate localizations of the nitroxide moieties of spin labels are indicated by arrows. Smaller 2A_Z values indicate higher hydrophobicity (upward changes in the profiles).

Figure 6

(A) The OTP obtained with ASL in cortical (dotted lines) and nuclear (solid lines) lens lipid membranes of eye lenses from human donors of different age groups. Data for age groups 41–60 and 61–70 years are reproduced, respectively, from Reference , Copyright 2013, with permission from Elsevier, and from Reference , Copyright 2014, with permission from Springer Publishing. (B) The same data as in (A) but plotted as a function of the Chol/PL mixing ratio in the suspension of lens lipid membranes. Notice that above the Chol/PL mixing ratio of ~2, the Chol crystals are formed in liposome suspensions. Thus, the Chol/PL molar ratio in PCDs and the amount of Chol forming CBDs should not increase further. Chol crystals form presumably outside the membrane and can be detected by the DSC only after a significant amount of Chol is accumulated in these Chol structures. In our investigations, Chol crystals were detected only for nuclear membranes from the age group 61–70 years with the Chol/PL molar ratio of 4.4. For nuclear membranes from the age group 41–60 years with the Chol/PL molar ratio of 2.1 we were unable to detect Chol crystals.

Figure 7

(A) The NiEDDA accessibility parameter obtained with CSL in cortical (dotted lines) and nuclear (solid lines) lens lipid membranes of eye lenses from human donors of different age groups. Data for age groups 41–60 and 61–70 years are reproduced, respectively, from Reference , Copyright 2013, with permission from Elsevier, and from Reference , Copyright 2014, with permission from Springer Publishing. (B) The same data as in (A) but plotted as a function of the Chol/PL mixing ratio in the suspension of lens lipid membranes. Notice that above the Chol/PL mixing ratio of ~2, the Chol crystals are formed in liposome suspensions. Thus, the Chol/PL molar ratio in PCDs and the amount of Chol forming CBDs should not increase further.

Figure 8

Schematic drawing presenting changes in the organization of cortical (inside broken rectangle) and nuclear lens lipid membranes (inside solid rectangle) as functions of age and Chol content in investigated membranes. Excess Chol (above the Chol solubility threshold at ~2) forms Chol crystals, presumably outside the fiber cell membranes. Notice that with age and Chol content, the Chol/PL molar ratio in PCDs and the size of CBDs change. The shading of domains indicates changes in measured domains properties. Increased shading of CBDs indicates that measured properties (OTP (Fig. 6) and NIEDDA accessibility parameter (Fig. 7)) change from those similar to properties of the PCD. (Insert) An expanded drawing of the lens lipid membrane with indicated membrane domains.