Measurement of lens protein aggregation in vivo using dynamic light scattering in a guinea pig/UVA model for nuclear cataract

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.     

Calcification of intraocular implant lens surfaces

Calcification of octacalcium phosphate [Ca8H2(PO4)6 x 5H2O, OCP] on differently packaged "Ultem" and "Surefold" intraocular implant lens surfaces has been studied in vitro in solutions supersaturated with respect to OCP at pH = 7.10 and 37 degrees C. No mineral deposition was observed on the lenses packaged in Ultem vials even after treatment with behenic acid, one of the fatty acids identified on explanted lenses. Following treatment with behenic acid, nucleation of OCP occurred on the lenses from Surefold vials, which incorporate silicone gaskets; induction periods preceding calcification were about 6 h. No mineralization was found on the lenses in vials with other gasket materials, including polytetrafluoroethylene, fluorocarbon elastomer, and polypropylene. The results of this study indicate that both silicone and fatty acids such as behenic acid play important roles in inducing the in vivo calcification of OCP on IOL lenses; all of the lens treatment steps were necessary for nucleation induction.     

Fatty Acids Profiling and Biomarker Identification in Snow Alga Chlamydomonas Nivalis by NaCl Stress Using GC/MS and Multivariate Statistical Analysis

The snow alga Chlamydomonas nivalis is a model species of microalgae for the investigation of cell response mechanism and adaptation ability in natural habitats in polar regions and similar extreme environments. The alteration of fatty acids in cellular lipids is known to play a vital role for cell survival and reproduction under various stress conditions. In the present work, an integrated approach of gas chromatography/mass spectrometry (GC/MS) coupled with multivariate statistical analysis was developed to investigate the fatty acid profiles and identify the biomarkers in response to NaCl stress. The data of fatty acid profiles between the control and NaCl-stress group was classified by orthogonal projection on latent structure discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA); six of fatty acids (C16:0, C16:3, C18:0, C18:1, C18:2, and C18:3) were identified as biomarkers. These biomarkers showed a regulatory role by decreasing the degree of lipid unsaturation (DLU), providing an expected function in reducing membrane fluidity and permeability for enhancing the tolerance to higher salinity. This is the first report to demonstrate the fatty acid biomarkers in microalgae as the physiological regulators corresponding to the response and adaptation to NaCl stress based on an integrated approach at the lipidomic level.     

Methods for extracting biochemical information from bacterial Raman spectra: focus on a group of structurally similar biomolecules--fatty acids

In this paper we explore the possibilities of Raman spectroscopy in order to deduce information on the fatty acid composition of bacterial cells. Therefore, representative strains of two bacterial taxa were each cultured in different conditions and in parallel analyzed by Raman spectroscopy and gaschromatographic FAME analysis. Raman spectra of pure fatty acids were recorded and used as reference spectra. The culturing conditions for each strain could be easily distinguished by the fatty acid information retrieved from bacterial Raman spectra. Chemometric techniques such as EMSC and PCA allowed to extract information about groups of fatty acids, that was consistent with the results from FAME analysis. Although the information retrieved from Raman spectroscopy is not as refined as that from FAME analysis, the presented methods could be useful to obtain basic information on the fatty acid present in bacteria when performing Raman spectroscopic analysis for fast whole cell profiling, which provides information for different types of cell components (fatty acids, amino acids, primary metabolites, etc.).     

Fatty acid profiling of raw human plasma and whole blood using direct thermal desorption combined with gas chromatography-mass spectrometry

Gas chromatography (GC) has in recent times become an important tool for the fatty acid profiling of human blood and plasma. An at-line procedure used in the fatty acid profiling of whole/intact aquatic micro-organisms without any sample preparation was adapted for this work. A direct thermal desorption (DTD) interface was used to profile the fatty acid composition of human plasma and whole human blood of eight volunteers in a procedure omitting the usual lipid extraction steps that precede sample methylation in the traditional (off-line) protocols. Trimethylsulfonium hydroxide (TMSH) was used as reagent for thermally assisted methylation. In a fully automated manner, the liner of the GC injector is used as a sample-and-reaction container with the aid of the DTD interface. The fatty acid methyl ester (FAME) profiles obtained using this novel approach, were very identical to those obtained when the traditional off-line protocol was applied. FAME yields obtained in the at-line DTD method were found to be very similar for saturated fatty acids, but significantly higher for polyunsaturated fatty acids compared to off-line yields. As a result of the contribution of circulating cell membranes in blood, substantial differences were observed when the amount of FAMEs obtained in whole human blood and human plasma samples were compared after their analysis. Thanks to the fully automated operation of this novel procedure, large series of analyses can easily be performed.     

Contribution on the characterization of lipids from the lenses of selected mammals

Total lipids from the lenses of pigs, calves, cattle and fin whales have been separated into different fractions. Their lipid content seems to be the lowest of all organs. The predominant lipid classes are cholesterol, ethanolamine cephalines, and sphingomyelines. The analysis of the fatty acids after saponification showed differences in the main components depending on the source of the material. In all cases 5–7% hydrocarbons were found which are probably of alimentary origin and are deposited in the lens.     

Evaluation of a rapid method for preparation of fatty acid methyl esters for analysis by gas-liquid chromatography

The major limitation to fatty acid analysis by gas-liquid chromatography is associated with preparation of fatty acid methyl esters (FAME). In the present study, FAME preparations were made from plant oils (corn, olive, sunflower), sunflower oil margarine, lard and various animal tissue fats by a rapid transesterification involving tetramethylammonium hydroxide in methanol, and also by a longer conventional saponification-esterification method. Fats from animal (beef, mutton, pork) adipose tissues were extracted by a simpler modified procedure and also by the Folch method prior to the rapid and the conventional FAME preparations, respectively. FAME analysis on a gas-liquid chromatograph equipped with a Silar 10C glass capillary column indicated similar fatty acid composition of a given fat or oil, whether FAME was prepared by the rapid or the longer conventional method. The data obtained by both methods were very highly correlated for all the fats (r = 0.9895 - 0.9999). However, the rapid method showed a tendency for enhanced recoveries of lower chain fatty acids (e.g. 14:0), and also of unsaturated C18 isomers. Possibly, losses of fatty acids that occurred during the lengthy fat extraction, fatty acid esterification or ether-evaporation FAME concentration steps (conventional method) were minimised by the single transesterification step (rapid method). This rapid transesterification method appears to be an attractive alternative to FAME preparation from a wide variety of different fats for gas-liquid chromatographic analysis.     

MEMBRANE DAMAGE IN UV-IRRADIATED LENSES

Abstract The purpose of this study was to investigate three possible causes of membrane damage following UV irradiation: photooxidation of membrane thiol (SH) groups, peroxidation of membrane lipids and inhibited synthesis of membrane proteins. Thiol loss was not observed. Thin-layer chromatography showed a four-fold increase in several primary lipid peroxidation products such as hydroperoxyl lipids in the epithelial membrane preparations isolated from irradiated lenses. The formation of new hydroxyl lipid bands not seen in control preparations was also observed in isolated membranes from irradiated lenses. Irradiation in the presence or absence of oxygen produced lipid peroxidation products. Aerobic irradiation produced small, but statistically significant increases in lipid hydroxyls and hydroperoxyls relative to controls. Repair of initial damage might be compromised by the observed 60% reduction in rate of protein synthesis measured in lens membranes following irradiation. Synthesis was affected by means other than depleted potassium or elevated calcium levels.     

Focal length variability and protein leakage as tools for measuring photooxidative damage to the lens

Hypericin is the ingredient used to standardize the popular over-the-counter antidepressant medication St. John's Wort. Because hypericin readily produces singlet oxygen and other excited state intermediates, it is a very efficient phototoxic agent in the eye that can potentially induce the development of the cataract photooxidative mechanism. Hypericin absorbs in the UV and visible ranges, binds to the lens crystallins (alpha, beta and gamma) and damages these proteins through a photooxidative mechanism. Effects were measured previously using fluorescence, UV and mass spectrometry. We report here two additional methods to monitor lens damage: (1) measuring focal length variability using a ScanTox instrument and (2) measuring protein leakage from the damaged lens. Because nonenzymic glycation results in free radical production, we chose to use elevated glucose concentrations as a convenient model for studying oxidative stress. To compare and contrast photooxidative damage against oxidative damage to the lens, we also measured the focal length variability and protein leakage induced by the presence of elevated glucose concentrations. We found that the total accumulated protein leakage was positively correlated (r = 0.9) with variability in focal length. Lenses treated with hypericin and irradiated with UVB had an increase in focal length variability as compared with the lenses that were only UVB-irradiated. Lenses without UVB irradiation had much lower focal length variability than irradiated lenses. For non-hypericin-treated lenses, UVB-irradiated lenses had a larger variability (4.58 mm) than the unirradiated lenses (1.78 mm). The lenses incubated in elevated glucose concentrations had a focal length variability (3.23 mm) equivalent to that of the unirradiated hypericin-treated lenses (3.54 mm). We conclude that photooxidative damage by hypericin results in changes in the optical properties of the lens, protein leakage and finally cataract formation. In contrast to this, high concentrations of glucose induced protein leakage but not changes in optical properties or the opacity associated with a cataract. This work provides further evidence that people should protect their eyes from intense sunlight when taking St. John's Wort.     

Fast analysis by gas-liquid chromatography. Perspective on the resolution of complex fatty acid compositions

Separation of fatty acids as methyl ester (FAME) derivatives has been carried out using short and highly polar capillary column developed for fast gas-liquid chromatography (GLC) applications. The GLC parameters have been optimized in order to achieve separation of FAME ranging from 4:0 (butyric acid) to 24:1 in less than 5 min. Milk fat that has by far the most complex fatty acid composition among edible fats and oils has been used to optimize the method. The volume of the oven has been reduced in order to allow for a heating rate of 120 degrees C/min and to rapidly cool-down to the initial temperature (50 degrees C) of the GLC program. The GLC conditions developed are not suitable to achieve separation of positional and geometrical isomers of octadecenoic acid but are useful to perform separation of major fatty acids in milk fat. The conditions developed could be used to analyze edible fats and oils or biological samples such as plasma or red blood cell lipids. The results confirmed that short and highly polar fast columns operating under optimal conditions could be used to separate the fatty acids in various matrices.     

Pyrolysis-mass spectrometry and gas chromatography-flame ionization detection as complementary tools for soil lipid characterization

Lipid biomarker profiles are a powerful tool for assessing soil microbial community structure, but intensive laboratory work and data analysis are needed to construct profiles from phospholipid fatty acids and other common biomarkers. Pyrolysis mass spectrometry (Py-MS) is a alternative method that provides a rapid and sensitive ‘fingerprint’ of soil lipids and may be sufficient to characterize lipids from various sites. The objective of this work was to evaluate the capacity of pyrolysis metastable atom bombardment time-of-flight mass spectrometry (Py-MAB-TOF-MS) to provide replicable analysis of soil lipids, compared to a routine gas chromatography-flame ionization detection (GC-FID) method. Soils were collected from six agricultural fields under soybean, corn and asparagus production. Soil lipids extracted with 1:2 chloroform:methanol solvent were analyzed with Py-MAB-TOF-MS or transesterified into fatty acids and then analyzed by GC-FID. The two methods were complementary, but distinct: lipid fingerprints, generated from Py-MAB-TOF-MS spectra, included extractable soil lipids from microbial, animal and plant origins plus non-living organic matter in the samples, whereas fatty acid profiles generally represented lipids from soil bacteria and fungi. We conclude that the soil lipid fingerprints generated from Py-MAB-TOF-MS present more variability than lipid biomarker profiles from the GC-FID method because they include a broader group of extractable soil lipids. Further work is needed to identify the molecular fragment masses in Py-MAB-TOF-MS spectra that could precisely identify soil lipids of microbial origin.     

Profiling eicosanoids and phospholipids using LC-MS/MS: principles and recent applications

Eicosanoids are potent lipid mediators involved in numerous physiological and pathophysiological processes. Precursors are polyunsaturated fatty acids liberated from membrane phospholipids. Thus, profiling and quantification of these molecules has gained a lot of attention during last years. Eicosanoids and phospholipids are commonly profiled by LC-MS/MSbecause this technique allows accurate quantification within acceptable run-times. This article therefore focuses on liquid chromatography and the ESI-MS/MS analysis of proinflammatory lipid mediators, particularly arachidonic acid (C20:4) derived eicosanoids and their precursors phospholipids. Recent analytical developments for quantification of these compounds are highlighted and analytical challenges are discussed. Furthermore, applications such as the use of these molecules as biomarkers are presented.     

Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by <Emphasis Type="Italic">in situ</Emphasis> transesterification

In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4–7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process.     

The direct determination of double bond positions in lipid mixtures by liquid chromatography/in-line ozonolysis/mass spectrometry

The direct determination of double bond positions in unsaturated lipids using in-line ozonolysis-mass spectrometry (O₃-MS) is described. In this experiment, ozone penetrates through the semi-permeable Teflon AF-2400 tubing containing a flow of a solution of fatty acid methyl esters (FAME). Unsaturated FAME are thus oxidized by the ozone and cleaved at the double bond positions. The ozonolysis products then flow directly into the atmospheric pressure photoionization (APPI) source of a mass spectrometer for analysis. Aldehyde products retaining the methyl ester group are indicative of the double bond positions in unsaturated FAME. For the first time, O₃-MS is able to couple directly to high performance liquid chromatography (HPLC), making the double bond localization in lipid mixtures possible. The application of LC/O₃-MS has been demonstrated for a fat sample from bovine adipose tissue. A total of 9 unsaturated FAME including 6 positional isomers were identified unambiguously, without comparison to standards. The in-line ozonolysis reaction apparatus is applicable to most mass spectrometers without instrumental modification; it is also directly compatible with various LC columns. The LC/O₃-MS method described here is thus a practical, versatile and easy to use new approach to the direct determination of double bond positions in lipids, even in complex mixtures.     

Fatty Acid Profiling During Microbial Lipid Production Under Varying pO2 and Impeller Tip Speeds

The fatty acid profile study was undertaken to study the effect of impeller tip speed-associated shear stress and dissolved oxygen saturation (DO) on the fatty acid composition variation and on total lipid content of the cells. The study was undertaken in a 5-l stirred tank bioreactor using Mucor sp. RRL001. To study the interaction of parameters and their effects, a central composite design was used. The fatty acid profiling during the course of study suggested that oleic acid and palmitic acid were two major components with their composition varying between 34-47% and 29-39.1%, respectively, of the total lipid content. The GLA content varied between 3% and 9% of the total lipid. The lipid profile study also revealed the presence of a minor amount of fatty acids of chain length C:12, C:20, C:22, and C:24. The modeling of lipid accumulation suggested that it follows a quadratic model with both impeller tip speed (p = 0.0166) and dissolved oxygen concentration (p = 0.0098) following the quadratic order of effect. The fermenter run based on the optimum production zone in response surface plot resulted in the maximum 4.8 g l(-1) lipid compared with the model-predicted value of 4.49 g l(-1). The present study suggests that dissolved oxygen saturation is a more significant contributor to total lipid accumulation. However, the study also suggests that the fatty acid profile of fungal lipid is not directly associated with the shear stress or oxygen availability in Mucor sp. RRL001.     

UVA Light‐mediated Ascorbate Oxidation in Human Lenses

Whether ascorbate oxidation is promoted by UVA light in human lenses and whether this process is influenced by age and GSH levels are not known. In this study, we used paired lenses from human donors. One lens of each pair was exposed to UVA light, whereas the other lens was kept in the dark for the same period of time as the control. Using LC‐MS/MS analyses, we found that older lenses (41–73 years) were more susceptible to UVA‐induced ascorbate oxidation than younger lenses (18–40 years). Approximately 36% of the ascorbate (relative to control) was oxidized in older lenses compared to ~16% in younger lenses. Furthermore, lenses with higher levels of GSH were less susceptible to UVA‐induced ascorbate oxidation compared to those with lower levels, and this effect was not dependent on age. The oxidation of ascorbate led to elevated levels of reactive α‐dicarbonyl compounds. In summary, our study showed that UVA light exposure leads to ascorbate oxidation in human lenses and that such oxidation is more pronounced in aged lenses and is inversely related to GSH levels. Our findings suggest that UVA light exposure could lead to protein aggregation through ascorbate oxidation in human lenses.     

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.     

Integrated multidimensional and comprehensive 2D GC analysis of fatty acid methyl esters

Fatty acid methyl ester (FAME) profiling in complex fish oil and milk fat samples was studied using integrated comprehensive 2D GC (GC × GC) and multidimensional GC (MDGC). Using GC × GC, FAME compounds – cis‐ and trans‐isomers, and essential fatty acid isomers – ranging from C18 to C22 in fish oil and C18 in milk fat were clearly displayed in contour plot format according to structural properties and patterns, further identified based on authentic standards. Incompletely resolved regions were subjected to MDGC, with Cn (n = 18, 20) zones transferred to a ²D column. Elution behavior of C18 FAME on various ²D column phases (ionic liquids IL111, IL100, IL76, and modified PEG) was evaluated. Individual isolated Cn zones demonstrated about four‐fold increased peak capacities. The IL100 provided superior separation, good peak shape, and utilization of elution space. For milk fat‐derived FAME, the ²D chromatogram revealed at least three peaks corresponding to C18:1, more than six peaks for cis/trans‐C18:2 isomers, and two peaks for C18:3. More than 17 peaks were obtained for the C20 region of fish oil‐derived FAMEs using MDGC, compared with ten peaks using GC × GC. The MDGC strategy is useful for improved FAME isomer separation and confirmation.     

Effect of Solvent on the Extraction of Microalgae Lipid for Biodiesel Production

The effect of solvent on the microalgae lipid extraction was studied. The efficiency of lipid extraction from microalgae was found to differ according to the solvent used. The existence of formic acid contributed to the extraction of lipid to a great extent. With 8 mg/L formic acid existing in the system, the lipid yield and free fatty methyl ester(FAME) yield increased from 39% to 42% and from 81% to 90% respectively compared to those of the control. The highest lipid yield of 42% was achieved from Chlorella protothecoidesis with an FAME yield of 89% when a mixed solvent of 14 mL/g dichloromethane, 2 mL/g methanol and 4 mL/g formic acid was used.     

Glycation and insolubility of human lens protein.

To learn whether glycation plays a role in insolubilization or in senile cataractogenesis, the reactivity of lens protein from normal and senile cataractous lenses and individual crystallin prepared from human lens with various sugars [glucose, glucose-1-phosphate (G-1-P), glucose-6-phosphate (G-6-P) and fructose], and the insolubility of those proteins were determined. The reactivity of human lens protein to glucose was increased in a dose-dependent manner, and it was demonstrated that 17.9, 18.5 and 24 kDa proteins were susceptible to glycation with sugars. The study also showed that alpha-, beta-crystallins and high molecular weight (HMW) aggregate obtained from cataractous lens have some weak reactivity against sugars. It was demonstrated that the proteins obtained from normal lens of older age and from cataractous lenses have higher insolubilities to glucose than do normal younger ones. Measurement of glycosylated protein by affinity column chromatography revealed that cataractous lenses contained a larger amount of glycosylated protein than normal ones. These results suggest that there is an age-related increase of glycation in normal human lens protein, and that such glycation increases the amount of insolubilized protein with the effect of aging. The author also speculates that an abnormal acceleration of glycation in the human lens may induce senile cataract formation.