Om P. Lamba

Spectroscopic detection of lipid peroxidation products and structural changes in a sphingomyelin model system

The peroxidation induced by tert-butyl hydroperoxide in sphingomyelin from bovine brain was investigated in detail. The lipid peroxidation products resulting from oxidation of lipid acyl chains were detected, identified and characterized by optical absorption. Fourier transform infrared, fluorescence and NMR spectroscopies. The extent of hydrocarbon chain degradation in vitro was quantified by measuring the relative change in absorbance of the peak at 241 nm characteristic of conjugated double bond or diene absorption band. FTIR data revealed that the lipid peroxidation of sphingomyelin disrupted the acyl chain and head group regions resulting in derangement of the ordered membrane.

Estimation of the secondary structure and conformation of bovine lens crystallins by infrared spectroscopy: quantitative analysis and resolution by Fourier self-deconvolution and curve fit

The secondary structure of six bovine lens protein fractions (two alpha, three beta and one gamma-crystallin) are examined in solution and in solid forms for the first time using FTIR spectroscopy. Films of the nuclear and cortical regions of the bovine lens are also examined. The structure is quantitatively estimated from the vibrational analysis of the resolution-enhanced amide-I profile achieved by Fourier self-deconvolution and linear least-squares curve-fit algorithm. All the protein fractions fold predominantly in a beta-pleated sheet structure with little or no alpha-helical domains in solution or in lyophilized solid form. These proteins also retain their predominant beta-sheet conformation in the cellular phospholipid environment of the lens, in conformity with the structure obtained for all the mammalian species examined to date. Despite structural homology, vibrational data indicate subtle structural differences within each class of the crystallins probably due to presence of several minor substructures/subconformations. Substantial high amounts of turns (approx. 40%) observed in the beta-fractions may have a fundamental implication in stabilizing the tertiary structure of the uniquely folded-proteins vital for the transparency of the lens. These proteins in solid KBr-matrix undergo a major structural change, induced primarily by ionic interactions which refold them in a helical conformation. IR spectroscopy together with band-narrowing procedures has proven to be an effective tool to obtain structural information of proteins in solution, as solid substrates or in a complex biological tissue, such as ocular lens.

Spectral characterization of lipid peroxidation in rabbit lens membranes induced by hydrogen peroxide in the presence of Fe2+Fe3+ cations: A site-specific catalyzed oxidation

The role of free-radical-induced lipid peroxidation (LPO) in relation to lens opacity is investigated using Fourier transform infrared spectroscopy. Phospholipids extracted from nuclear and cortical regions of the rabbit lens membranes are subjected to oxidative-damage induced by hydrogen peroxide and Fe2+/Fe3+ cations. Vibrational data suggest a homolytic decomposition of the unsaturated membrane hydrocarbon chains at cis-double bonds, as well as structural modifications at the carbonyl and phosphate-oxygen sites of the fiber cell membranes upon metal oxidation. This is also evident from a substantial induction of the carbonyl groups and a significant dephosphorylation of the phosphate groups in lens phospholipids. These covalent modifications and/or alterations of the carbonyl and phosphate groups, and specificity of certain vibrational modes only to iron oxidation, may serve as a diagnostic probe of the metal-catalyzed LPO in lens membranes. Despite covalent modifications of the hydrophilic part of the lens membranes, hydrocarbon chain region remains largely intact at physiological concentrations of hydrogen peroxide. However, at elevated concentrations of hydrogen peroxide, a substantial breakdown of the acyl chains occurs. Striking similarities observed between the spectral features of the oxidized rabbit lens phospholipids and those of the cataractous human lenses suggest that the mechanism and pathways of lipid oxidation in model animal membranes and in human lenses are similar. Differences in the nuclear or cortical regions are also evident upon metal oxidation. Nuclear lipids experience increased effects of the metal oxidation compared to cortical lipids. Both the nuclear or the cortical lipids indicate effective penetration of the bilayer water creating segregated membrane domains, possibly through breakdown of headgroup-specific lipid-water interactions. This could effectively alter the lens membrane permeability and fluidity, rendering it susceptible to a host of toxic oxidants present in the eye. These findings also demonstrate that LPO can lead to acyl chain degradation that may effectively derange the lens membrane function, which could be a contributing factor in cataractogenesis.

Raman structural characterization of clear human lens lipid membranes

Raman spectroscopy was used for the first time to characterize the structure of lipid membranes prepared from the nuclear and cortical regions of 48 and 69 year old clear human lenses. The interface region carbonyl band appears as a doublet at 1742 and 1728 cm-1. The lower frequency band is characteristic of a hydrogen bonded carbonyl group, perhaps to bilayer water. From the intensity of the curve fit bands, we calculate that 43% of the carbonyl groups are hydrogen bonded. Our data show that the hydrocarbon chains of the nuclear lipids are 1.4 times more saturated than those of the cortical lipids. The molar ratio of phospholipid CH2/= C-H groups was calculated to be 13 and 18 for cortical and nuclear lipids, respectively. Hydrocarbon chain disorder was estimated to be 72 and 58% (+/- 8% disorder) for the cortical and the nuclear lipids, respectively. Raman spectroscopy is sensitive to structural differences in various regions of the lipid bilayer and could be an effective tool to explore lipid and protein interactions in terms of lens region, age and opacity.

Structural characterization of clear human lens lipid membranes by near-infrared Fourier transform Raman spectroscopy

Regional differences in human lens membrane lipid composition have been documented and could be responsible for alterations in the function of lens membranes. The phospholipid composition of epithelial membranes of human lenses has been shown to be different from that of fiber membranes. To establish lipid composition-membrane structure relationships, we have examined spectroscopically the structure of lipid membranes from human lens epithelium, cortex and nucleus. Near-infrared Fourier transform Raman spectroscopy was used to obtain the lipid structure of membranes in which the lipid composition was determined previously by 31P-NMR. The disorder (fluidity measured structurally) of the epithelium was evaluated to be 80%, whereas that of the lipids from the cortical and nuclear regions was 55%. The large size of the band at 1650 cm-1 arising from sphingolipids supported the compositional studies which indicate that the major component of human lens membranes is a sphingolipid. Sphingolipids probably account for the high degree of lipid order found in lens membranes. Epithelial membranes were found to contain more glycerolipids and less sphingolipids than fiber cell membranes. This compositional difference would be expected to disorder the epithelial membrane.

Spectroscopic and Related Studies on Lens Epithelial Lipids

In an effort to understand the regional lipid structural and compositional differences which occur in clear and cataractous human lenses we characterized the structure of lipid membranes using infrared (1, 2) and Raman (3) spectroscopy. Significant regional differences were found between the membrane structure of lipids from the cortical and nuclear regions as well as between clear and cataractous lenses (3). It was proposed that alterations in lipid composition and structure could play a role in cataractogenesis.