Biomedical subjects

L T Chylack

Publications and source records attributed to L T Chylack.

At least 91 records · Page 5Linked to original sources

PubMed1986

Acute glucose-derived osmotic stress in rabbit lenses.

The morphological changes observed in rabbit lenses incubated without corpus vitreum in medium whose glucose concentration varied cyclically between normoglycaemia and hyperglycaemia were analyzed for the location of light scattering vacuoles. Minimal changes were seen in the epithelial cells, the external bow fibres and the lens nucleus, while maximal changes with vacuolization and damage were seen in the posterior part of the inner bow fibres. It was shown that the exchange of water takes place mainly across the posterior capsule. The susceptibility of this region in part may be due to the low aldose reductase activity in this region of the lens.

Animals↗

PubMed1986

Sorbitol production in the lens: a means of counteracting glucose-derived osmotic stress.

Heretofore, the intracellular accumulation of sorbitol has been associated exclusively with deleterious (cataractogenic) changes in the lens. This study demonstrates a beneficial role for the sorbitol pathway in the rabbit lens, namely that of counteracting extracellular, glucose-derived, osmotic stress with the intracellular production of osmotically active sorbitol. Large and sudden increases in the extracellular glucose concentration lead to dehydration of the lens, a response that can be diminished by intracellular sorbitol and fructose production. These results are discussed in light of the impact (beneficial/detrimental) of aldose reductase inhibitors on the lens. Sugar cataract formation appears to result from continuous, rather than cyclical, activity of a pathway which normally may have a protective function in the lens.

Animals↗

PubMed1986

The effect of oxidation on sorbitol pathway kinetics.

The rapid conversion of glucose to sorbitol by aldose reductase and the consequent hyperosmolarity of the cytoplasm has been shown to be the primary cause of the so-called "sugar" or "osmotic" cataract in many animal lenses. It is not as clear, however, that hyperosmolarity is the principal factor in the etiology of cataracts in human diabetic subjects. In fact, the comparatively low activity of aldose reductase in the human lens as compared with several animal lenses, and the osmotically insignificant levels of sorbitol pathway products (sorbitol and fructose), suggest that hyperosmolarity, per se, may not be as important a factor in human cataract formation as it is in animals. We present evidence that the flux of glucose and sorbitol through the rat lens is markedly reduced by oxidative stress (0.1 mM H2O2). Sorbitol accumulation is reduced by 114%, sorbitol turnover is reduced by 78%, sorbitol production is reduced by 90%, fructose accumulation is reduced by 60%, and fructose turnover is reduced by 76% in the presence of 36 mM glucose. H2O2 does not affect glucose turnover, the glucose rate constant, or the ATP level significantly at 36 mM glucose, but at 5.5 mM glucose, 0.2 mM H2O2 leads to a rapid loss of ATP that can be prevented by 0.04 mM sorbinil, an aldose reductase inhibitor. These results suggest that inhibition of aldose reductase by sorbinil renders rat lenses better able to cope with oxidative stress. In the absence of an aldose reductase inhibitor, elevating ambient glucose may render a lens less able to scavenge oxidants by diverting NADPH into sorbitol production.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1986

Topographic correspondence between total and non-freezable water content and the appearance of cataract in human lenses.

Eight intracapsular and 18 extracapsular human surgical specimens were stereophotographed. Each lens was divided into 10 areas, separating those which appeared transparent from those that showed opacities. Samples weighing 6-12 mg from each area were investigated. The total water content was determined by thermogravimetric analysis; the freezable water content was measured by differential scanning calorimetry. The difference between the two provided the non-freezable water content. Graphic presentation illustrates the correspondence of high total water, low non-freezable water content with location of the turbidity in the lens. Pairwise, statistical comparison shows that in intracapsular human surgical specimens the non-freezable water content in the clear areas of both cortex and nucleus was significantly greater than in the opaque areas.

Body Water↗

PubMed1986

In vivo measurement of the aging rabbit lens using quasielastic light scattering.

We have been able to analyze the autocorrelation function of the light scattered from the rabbit lens in vivo in terms of a two component, exponential fit. We have measured the intensity and the decay rate associated with each of these two components as a function of age and position in the lens. As far as is possible we have interpreted these results in terms of molecular changes in the state of association of the protein constituents in the aging rabbit lens. These studies suggest that the method of quasielastic light scattering spectroscopy can provide a useful probe of the protein modifications that occur in both normal and cataractous human lenses.

Aging↗

PubMed1986

The protective effect of glucose on soluble rat lens hexokinase in the presence of oxidative stress.

An in vitro animal model was used to characterize the protective effect of glucose on lenses subjected to oxidative stress. Paired rat lenses were incubated in TC-199 medium for six hours in the presence of an oxidant (0.06 mM H2O2, superoxide produced from 5 mM purine, or hydroxyl radical) and 2 mM glucose (control) or no glucose (experimental). Soluble hexokinase (HK) specific activity and lactate production were measured. 0.06 mM H2O2 inactivates 48% of the hexokinase in the absence of glucose; with glucose present hexokinase activity is reduced only 26%. Control experiments without oxidants show a statistically insignificant difference between hexokinase activities in the 0 and 2 mM groups, suggesting that the changes observed are not simply due to the presence or absence of glucose. Hexosemonophosphate shunt activity increases nearly 2.5-fold in the presence of 0.06 mM H2O2 and 2.0, 4.0 or 5.5 mM glucose. This suggests that the loss of hexokinase (a -SH enzyme) in the presence of H2O2 and 0 mM glucose is due to NADPH production inadequate to offset the oxidative stress on enzyme -SH groups. FPLC analysis suggests that type II HK is more susceptible to oxidative inactivation than type I, and further studies have shown that this inactivation is localized to the capsule/epithelium. Lactate levels were measured and controls (without oxidants) were run, to obtain a baseline value for fresh lenses and assess the contribution of endogenous glucose to lactate production. H2O2 levels in superoxide and hydroxyl radical media were measured, and the protective effects of mannitol and catalase were also determined.

Animals↗

PubMed1986

Inexpensive stereoscopic CCRG camera for lens/cataract photography in vitro.

In 1978 the American Cooperative Cataract Research Group (CCRG) adopted a stereoscopic camera system for photographing human cataracts in vitro based upon a Zeiss OPMI 1 operating microscope (Carl Zeiss, Inc.; Oberkochen, West Germany). Photographs obtained with this system were used to classify human cataractous change according to the standardized CCRG protocol. Classification data correlated with laboratory data furthered the attempt to define the biochemical or biophysical basis for specific types of cataractous change. Presently, the high cost of this camera (exceeding $20,000) precludes its use by many laboratories wishing to do human lens research. This study describes an inexpensive (less than $2,200) alternative camera for this type of photography. Adjacent frames on the film strip constitute stereo pairs which can be viewed in a modified stereo viewer. In the original CCRG camera both members of a stereo pair were included in the same frame. The quality of the stereo images obtained with this new system nearly equals that with the original Zeiss system. It is hoped that this inexpensive system will allow more scientists to participate in CCRG-related research and increase the supply of intracapsularly extracted cataracts available to all collaborating CCRG scientists.

Cataract↗

PubMed1986

Freezable and non-freezable water content of cataractous human lenses.

Twenty-nine cataractous human lenses between the ages of 36 and 93 were investigated. The total water content of cortex and nucleus were obtained by vacuum dehydration. The freezable water content was measured by differential scanning calorimetry. The non-freezable water content was obtained by difference. To measure the extent that syneresis contributes to cataract formation, an excess function was designed. The excess function represents the changes that occur, for example, in non-freezable water content due to cataractogenesis. It is calculated as the difference between the non-freezable water content of a normal lens and that of a cataractous lens of the same age. On the average, there is 7% less non-freezable water in the cortex of a cataractous lens than in the cortex of a normal lens. The corresponding number for the nucleus is 9%. This represents 29 and 36% changes, respectively, in the cortex and nucleus due to syneresis during cataractogenesis.

Adult↗

PubMed1985

Spectroscopic studies on human lens crystallins.

Human lens crystallins were studied by absorption, circular dichroism and fluorescence spectroscopy. The absorption spectra in the near-ultraviolet region show some differences in intensity, but spectral features are similar, except for the alpha-crystallin, which gives a fine structure due to phenylalanine between 250 and 270 nm. Tryptophan fluorescence and near-ultraviolet circular dichroism indicate that tryptophan residues are more exposed in alpha-crystallin than in either beta- or gamma-crystallin, and that the degree of exposure decreases in the order of alpha less than beta 1 greater than beta 2 greater than beta 3 greater than gamma. The far ultraviolet CD suggests that these proteins exist mainly in a beta-sheet conformation and that the amount does not vary much among them. The greater exposure of the tryptophan residues in the high-molecular-weight crystallins may reflect greater unfolding in their protein domains. Spectroscopic measurements are thus useful in predicting protein tertiary structure in the absence of the complete sequence and X-ray data. The fact that the high-molecular-weight proteins exist in a more unfolded state may render them more vulnerable to exogeneous insults, and these effects may be studied by spectroscopic measurements.

Chromatography, Gel↗

PubMed1985

Sorbitol/fructose metabolism in the lens.

The function of the sorbitol pathway, both as a secondary energy source and as an osmotic counterbalancing force, was examined. Rat lenses were incubated in media containing fructose as the primary exogenous energy source, and 31P nuclear magnetic resonance (NMR) spectra were accumulated. Fructose was found to be a sub-optimal but usable substrate for glycolysis. The utilization of fructose was further confirmed by a 14C fructose tracer study, using high-pressure liquid chromatography. Thus the intralenticular pool of sorbitol + fructose could serve as a secondary energy source during severe hypoglycemia in the diabetic lens. However, fructose is not a physiologically significant alternative to glucose. 13C NMR spectroscopy was employed to determine the kinetics of sorbitol/fructose accumulation in lenses incubated in 35.5 mM 13C1-glucose, and the sorbitol/fructose consumption after the preincubated lenses were transferred to media containing no glucose. Based on these kinetic studies, we concluded that the sorbitol pathway cannot generate sorbitol/fructose fast enough to offset increased osmotic pressure from high glucose levels in the aqueous humor of the diabetic eye. The contribution of osmotic equivalents from sorbitol + fructose, however, cannot be ignored.

Adenosine Triphosphate↗

PubMed1985

Glucose metabolism in the calf lens.

Carbohydrate metabolism in lens homogenates was evaluated using uniformly labeled [14C]-glucose. Metabolites were separated using HPLC and the distribution of 14C among metabolic pathways was determined. Under the standard incubation conditions employed (pH 7.4, 37 degrees C, 5 mM glucose) 40% of the glucose consumed forms lactate. The amount of 14C found in sorbitol and alpha-glycerol phosphate is 1-2%. This pathway distribution is essentially independent of glucose concentration from 2 to 35 mM. Over this range, lactate production is relatively constant at 2-3 mumol 7 hr-1. At 35 mM glucose, sorbitol production increases to 0.182 from 0.036 mumol 7 hr-1 at 5 mM glucose. The level of ATP is clearly a very significant factor in the regulation of glycolytic rate. An increase in ATP concentration from 1 to 3 mM results in a decrease in the rate of lactate production from 2.09 to 0.404 mumol 7 hr-1. The NAD/NADH ratio is significant in the distribution between lactate and alpha-GP. An increase in the level of NADP or NADPH increases sorbitol production as well as alpha-GP production. A computer program was developed to model the experimental incubation system. The calculated results are in agreement with the experimental in showing effects of ATP and perturbations of enzyme cofactor concentrations.

Adenosine Triphosphate↗

PubMed1985

Light scattering of whole excised human cataractous lenses. Relationships between different light scattering parameters.

The light scattering intensities of 27 cataractous human lenses were obtained as a function of scattering angle. Unpolarized white light (tungsten) and polarized light of Hg lamp at 435.8 and 546.1 nm were used in the I parallel and I+ modes. Selected light scattering parameters such as relative scattering intensities (I45/I0), dissymmetry ratio (I135/I45) and depolarization ratios (I45/I0)+/(I45/I0) parallel were correlated with the age of lens with percent transmission using regression analysis. Excess scattering functions were calculated that quantitatively describe the quality of lens. These excess functions were also correlated with other scattering parameters. A method which can be used to estimate the light intensity that reaches the retina at different scattering angles for a cataractous lens once the back scatter intensity (slit lamp examination) is known is given.

Adult↗

PubMed1985

Validity and reproducibility of the Cooperative Cataract Research Group (CCRG) cataract classification system.

The validity and reproducibility with which six classifiers [one experienced (L.T.C.), and five novices (W.G., F.G., W.W., J.W. and O.W.)] used the CCRG cataract classification system was assessed. The validity of index classifications was assessed by computing sensitivities and pairwise interclass correlations between experienced and novice classifiers using the former's classification as the standard. The number of unordered combinations of terms in the CCRG's classification was reduced by combining cortical terms according to the CCRG's accepted system of staged simplification. The number of combinations of terms at each stage is as follows: Stage I (greater than 1000); II (127); III (63); IV (15); V (7); VI and VII (3) and VIII (2). Excellent agreement was obtained between the experienced and novice classifiers for Stages VII and VIII of the classification, good agreement for Stages V and VI and poor agreement for Stages IV, III and II (sensitivities of 97, 96, 72, 59, 40, 24 and 20% respectively). Good agreement was also achieved for the classifications of single lenticular regions, except for subcapsular regions. The intra- and interobserver reproducibility was assessed by computing the Kappa statistic to (1) compare classifications between novice observers and (2) compare repeat classifications made by the same observer by viewing the same cataract once on each of three different days. The novice classifiers had excellent intraobserver reproducibility for Stages VII and VIII (Kappas of 0.87 and 0.97 respectively), good reproducibility for Stages IV, V and VI (Kappas of 0.53, 0.62 and 0.62, respectively) and marginal reproducibility for stages II and III (Kappas of 0.39 and 0.40, respectively). The intraobserver reproducibility of the experienced classifier was superior to the others for virtually all characteristics with excellent reproducibility for Stages IV, V, VI, VII and VIII with Kappas of 0.79, 0.90, 1.0, 1.0 and 1.0, respectively and good reproducibility for Stages II and III (Kappas of 0.55 and 0.64, respectively). These results indicate that the simplified CCRG cataract classification system (Stages IV-VIII) passes the minimum standards for reproducibility. The performance of the experienced classifier far exceeds the minimum standards and indicates the feasibility of improving classifier performance with training and practice.

Cataract↗

PubMed1985

Fluorescence studies on tryptophan and sulfhydryl group changes of bovine lens crystallins in a photodynamic system.

Conformational changes in the three crystallins alpha-, beta-, and gamma- in a singlet-oxygen generating system were investigated by fluorescence studies of tryptophan and covalently-bound sulfhydryl probe 4-[(N-iodoacetoxy)N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD). Upon excitation at 295 nm, the tryptophan emission maxima of the crystallins were red-shifted by irradiation with visible light in the presence of the photosensitizer methylene blue. beta- crystallin showed the largest shift (4 nm) of the emission spectrum. Time course of the fluorescence changes by irradiation showed that the decrease in the tryptophan fluorescence yield occurs most rapidly for beta-crystallins, as compared to alpha- or gamma-crystallins. Fluorescence changes of IANBD-labeled crystallins show a 40% decrease in the fluorescence intensity of the sulfhydryl probe for beta-crystallin after one hour of irradiation. For alpha- and gamma-crystallin smaller decreases (7% and 15% respectively) were observed. Since all the sulfhydryl groups of beta-crystallin are known to be exposed on the surface of the protein (Andley et al, 1982, Biochemistry 21, 1853), these results suggest that the pronounced changes in conformation of beta-crystallin by singlet oxygen may be due to a rapid loss of the protein tertiary structure by oxidation of the sulfhydryl groups. These results have potential significance in understanding the age and cataract-related changes in the ocular lens in view of the fact that several key lens enzymes are associated with beta-crystallins in vivo.

Absorption↗

PubMed1984

Change in the protein tertiary structure with non-enzymatic glycosylation of calf alpha-crystallin.

Non-enzymatic glycosylation of calf alpha-crystallin was induced by incubation with glucose. Glycosylated and non-glycosylated proteins were separated by affinity chromatography on Glyco Gel B boronic acid and were studied by circular dichroism (CD) and fluorescence. CD indicated that the glycosylated protein secondary structure was not altered, but the tertiary structure did undergo some changes. The CD band of this protein between 290 and 310 nm decreased in intensity. Extrinsic fluorescence probes, TNS (6-(p-toluidinyl) naphthalene-2-sulfonate) and MIANS (6-(4'-maleimidyl-anilino) naphthalene-2-sulfonic acid), indicated changes in both TNS binding sites and the sulfhydryl groups to a less hydrophobic microenvironment. Our results suggest that the glycosylation of protein leads to partial unfolding, and facilitates the sulfhydryl to oxidation.

Animals↗

PubMed1984

Classification of human cataractous change by the American Cooperative Cataract Research Group method.

The American Cooperative Cataract Research Group (CCRG) has adopted a system of classifying cataractous changes in excised human cataracts that is based on separate and independent photographic documentation of opacification and nuclear colour. The classification data can be simplified according to the needs of the scientist in his or her effort to measure clinically or scientifically significant associations between laboratory measures and cataractous change. The association between nuclear colour and the extent of cortical opacification and the intensity of nuclear opacification has been studied and found to be insignificant. These results justify the recommendation that nuclear colour be abandoned as the single index of severity of any type of senile cataractous change. The adaptability of this system to in vivo use in epidemiological and other studies of the natural history of cataractous change is discussed. Its limitations are outlined. The system may offer a basis for international cooperation in cataract classification.

Cataract↗

PubMed1984

Altered phosphate metabolism in the intact rabbit lens under high glucose conditions and its prevention by an aldose reductase inhibitor.

Intact paired rabbit lenses were incubated in media containing 5.5 mM and 35.5 mM glucose (both at 290 +/- 3 mOsm) and examined by phosphorus-31 nuclear magnetic resonance spectroscopy. Lenses in 35.5 mM glucose exhibited an altered metabolic steady-state characterized by elevated alpha-glycerophosphate and depressed adenosine triphosphate concentrations. Time course studies revealed that these metabolic changes occurred chiefly during the initial 48 hr of incubation under high glucose conditions. The inclusion of an aldose reductase inhibitor in the medium prevented these changes in lenticular metabolism.

Adenosine Triphosphate↗

PubMed1984

Mechanisms of senile cataract formation.

Research on the mechanisms of lens opacification during the past 20 years has revealed a number of individual, identifiable cataractogenic stresses in man. They include osmotic cataract formation (diabetic, galactosemic and hypoglycemic cataracts), radiation cataracts (X-irradiation, near U.V. radiation and microwave radiation) and in senile cataract the conversion of soluble low molecular weight cytoplasmic proteins to soluble high molecular weight aggregates, insoluble phases, and insoluble membrane-protein matrices. Oxidative stress has emerged as a common denominator of many changes in senile cataract. As we increase our understanding of these mechanisms, we may be able to intervene therapeutically to delay or prevent human cataract formation in man.

Aged↗