Fatty acid uptake and incorporation into phospholipids in the rat lens

PURPOSE. Phospholipids are a major component of lens fiber cells and influence the activity of membrane proteins. Previous investigations of fatty acid uptake by the lens are limited. The purpose of the present study was thus to determine whether exogenous fatty acids could be taken up by the rat lens and incorporated into molecular phospholipids. METHODS. Lenses were incubated with fluorescently labeled palmitic acid and then analyzed by confocal microscopy. Concurrently, lenses incubated with either fluorescently labeled palmitic acid or the more physiologically relevant (13)C(18)-oleic acid were sectioned into nuclear and cortical regions and analyzed by highly sensitive and structurally selective electrospray ionization tandem mass spectrometry techniques. RESULTS. The detection of fluorescently labeled palmitic acid, even after 16 hours of incubation, was limited to approximately the outer 25% to 30% of the rat lens. Mass spectrometry also revealed the presence of free (13)C(18)-oleic acid in the cortex but not the nucleus. No evidence could be found for incorporation of fluorescently labeled palmitic acid into phospholipids; however, a low level of (13)C(18)-oleic acid incorporation into phosphatidylethanolamine (PE), specifically PE (PE 16:0/(13)C(18) 18:1) was detected in the lens cortex after 16 hours. CONCLUSIONS. These data demonstrate that uptake of exogenous (e.g., dietary fatty acids) by the lens and their incorporation into phospholipids is minimal, most likely occurring only during de novo synthesis in the outermost region of the lens. This finding adds support to the hypothesis that once synthesized there is no active remodeling or turnover of fiber cell phospholipids.

Phospholipid composition of the rat lens is independent of diet

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

UVB irradiation alters the activities and kinetic properties of the enzymes of energy metabolism in rat lens during aging

Ultraviolet (UV) radiation is one of the major risk factors of cataract (loss of eye-lens transparency). The influence of UVB radiation (300 nm, 100 mu W cm(-2)) on the activity and apparent kinetic constants (K-m and V-max) of rat lens hexokinase (HK;EC2.7.1.1), phosphofructokinase (PFK;EC2.7.1.11), isocitrate dehydrogenase (ICDH;EC1.1.1.41) and malate dehydrogenase (MDH;EC1.1.1.37) of energy metabolism has been investigated by irradiating the lens homogenate of three-and 12-month-old rats. In the three-month-old group specific activities of HK and PFK are reduced by 56 and 43 %, respectively, and there is no change in ICDH and MDH activities after a 24 h exposure. On the other hand, in the 12-month-old group the decreases are 72, 71, 24 and 16 % for HK, PFK. ICDH and MDH, respectively. UVB irradiation increases the apparent K-m of HK and PFK (in both age groups), whereas the K-m of ICDH and MDH is not altered. While the decrease in V-max of these enzymes due to UVB exposure is only marginal in three-month-old rats, it is more pronounced (significant) in 12-month-old rats. A similar decrease in enzyme activities of HK and PFK is also observe upon UVB exposure of the intact rat lens. The photoinduced changes in energy metabolism may in turn have a bearing on lens transparency, particularly at an older age.

Protection against UVB inactivation (in vitro) of rat lens enzymes by natural antioxidants

Oxidative damage, through increased production of free radicals, is believed to be involved in UV-induced cataractogenesis (eye lens opacification). The possibility of UVB radiation causing damage to important lenticular enzymes was assessed by irradiating 3 months old rat lenses (in RPMI-1640 medium) at 300 nm (100 mu Wcm(-2)) for 24 h, in the absence and presence of ascorbic acid, alpha-tocopherol acetate and beta-carotene. UVB irradiation resulted in decreased activities of hexokinase, glucose-6-phosphate dehydrogenase, aldose reductase, and Na, K- ATPase by 42, 40, 44 and 57% respectively. While endopeptidase activity (229%) and lipid peroxidation (156%) were increased, isocitrate dehydrogenase activity was not altered on irradiation. In the presence of externally added ascorbic acid, tocopherol and beta-carotene (separately) to the medium, the changes in enzyme activities (except endopeptidase) and increased lipid peroxidation, due to UVB exposure, were prevented. These results suggest that UVB radiation exerts oxidative damage on lens enzymes and antioxidants were protective against this damage.

Synergistic effect of UVB radiation and age on HMPS enzymes in rat lens homogenate

The behaviour of rat lenticular enzymes, glucose-6-phosphate dehydrogena.se (G6PD, EC: 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGD, EC: 1.1.1.44) as a function of age and UVB irradiation (in vitro) was investigated by irradiating the lens homogenate from 3-and 12-month-old rats at 300 nm (100 μW cm 2). In the 3-month-old group the specific activities of G6PD and 6PGD were reduced by 26% and 42%, respectively, after 24 h of irradiation, whereas in the 12-month-old group the decrease was 38% and 49% respectively, which suggests that the susceptibility of HMPS enzymes to UVB damage is higher in older lenses. The decrease in specitic activity was associated with a change in apparent Km and Vmax (marginal in 3 months and significant in 12 months) of these enzymes due to UVB irradiation. UVB irradiation also decreased the levels of NADPH and NADPH/NADP ratio. These changes, altered activities of G6PD and 6PGD and altered levels of NADPH. may in turn have a bearing on lens transparency.

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.

Electric currents and lens regeneration in the rat

We studied the process of lens regeneration in the rat following an extracapsular lens extraction preserving the anterior lens capsule and anterior lens epithelium. We assessed clinically the clarity of the newly regenerated lens, evaluated changes in the lens electrical currents following surgery and during the regeneration process and correlated these changes with findings on light microscopy. Protein analysis of the regenerated lens was also undertaken. Experiments were performed in 41 Sprague-Dawley rats, sacrificed at 0, 2, 4 and 8 weeks postoperatively. Our results showed that complete lens regeneration occurred 8 weeks postoperatively only if the anterior epithelium was preserved and the lens capsule was closed surgically. Lens electrical currents, altered following surgery, recovered in parallel with the process of regeneration of the lens. The newly regenerated lens was optically clear and biochemical analysis revealed a pattern of protein expression resembling that observed during lens development. In conclusion, complete lens regeneration occurs in the rat and it is possible that lens electrical signals, together with other cues, may play an important role in this process. © 2009 Elsevier Ltd.

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. © 2008 Elsevier B.V. All rights reserved.

Polyamine system in developing rat eye and an animal model of retinopathy of prematurity

BACKGROUND: In experimental models of retinopathy of prematurity (ROP), a vasoproliferative disorder of the retina, retinal lesions are usually assessed by morphological examination. However, studies suggest that the polyamine system may be useful in monitoring proliferation processes. For this reason, polyamine concentrations in rat erythrocytes (RBC) and the regulation of polyamine system in rat eyes under the conditions relevant to ROP were investigated. METHODS: Newborn Wistar rats were reared in room air (control) or exposed first to hyperoxia (60% or 80% oxygen, 2 weeks) and then to normoxia (relative hypoxia, 1 or 2 weeks). Blood was collected from orbital vessels at 2 weeks of age and before death. Polyamine system-related enzyme activities were measured in retina and lens with radioassays. Polyamines were quantified by fluorometry after extraction, dansylation and HPLC separation. RESULTS: Oxygen (80% only) significantly decreased RBC polyamine concentrations, which then markedly increased after rats were transferred for a week to normal air, suggesting retardation of growth processes and compensatory stimulation, respectively. However, polyamine system changes in the rat eye were not so pronounced. Enzyme activities and polyamine concentrations tended to be lower in retina after hyperoxia and were only slightly higher, with the exception of ornithine decarboxylase, after a subsequent 1 week of normoxia. In litters subjected to normoxia for longer periods no changes were found. CONCLUSION: The transient and short-lived alteration in polyamine metabolism, especially in the eye, suggests that exposure of newborn rats to high oxygen supplementation followed by normoxia does not necessarily result in marked retinopathy.

Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands.

The outset of mental torture. Through the lens of the Ticking Time Bomb Scenario

No doubt shall be placed when qualifying torture as one of the cruellest crime offences against human beings. It is widely known that the first torture practices go back to the Middle Ages, where torture mechanisms and devices were used as a legitimate means of punishment, extraction of confessions or executions. Brutal techniques such as ‘Judas Cradle’, ‘The Rack’ or the ‘Rat Torture’ were indeed, the ones commonly used. Moreover, some centuries onwards, torture warrants were permitted and authorised by Privy Councils in legislations such as the English one. However, examples like that were the only ones which public accountability was given to, whereas off-the-book practices remained in silence in other countries for long lasting years. Nowadays, in the 21st century, there are innumerable enforced laws and provisions that prohibit the act of torture, to be precise, physical and psychological torture. Nonetheless, not only are these legislations necessary for fighting torture, but also ad hoc courts and specialised committees continuously report the existence of this crime offence.