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Biomedical subjects

J R Trevithick

Publications and source records attributed to J R Trevithick.

At least 19 recordsLinked to original sources
PubMed2003

Optical function and mitochondrial metabolic properties in damage and recovery of bovine lens after in vitro carbonyl cyanide m-chlorophenylhydrazone treatment.

In order to elucidate the correlation between lens optical function and metabolic function, in vitro bovine lens optical quality and mitochondrial integrity was measured following treatment with carbonyl cyanide m-chlorophenylhydrazone (the mitochondrial depolarizing agent, CCCP). The results indicate that in vitro exposure to CCCP resulted in concentration and time-dependent loss of sharp focus. The concentrations tested included 65.0, 32.5, 16.25 and 8.125 microm CCCP. Lenses treated with two lower concentrations show recovery from damage at the 24-h scan point. In lenses treated with 65 microM CCCP, mitochondria in lens epithelial and superficial cortical fibre cells appeared short and swollen. The results of this study indicate that lens optical function and mitochondrial integrity are closely correlated.

Journal Article↗
PubMed1999

Modelling cortical cataractogenesis XXIV: uptake by the lens of glutathione injected into the rat.

PURPOSE: Work of several groups including ours has shown that injection of glutathione may help to prevent the formation of cataract in the rat lens both in vitro and in vivo. These experiments were initiated to investigate the mechanism by which injected glutathione reaches the lens in vivo. The route is uncertain, but might involve either aqueous or vitreous humors, in contact with the lens anterior and posterior, respectively. Kannan's work has indicated that glutathione can be taken up ex vivo from the aqueous, by perfused isolated lens, but has not investigated; (1) whole animal glutathione injections, (2) the relative proportion of reduced and oxidized glutathione, and (3) the possibility that uptake can occur from the vitreous (in contrast to the aqueous humor) route. METHODS: 3H- or 35S-glutathione was injected into rats intraperitoneally and the radioactivity in serum and lens homogenates followed. RESULTS: The 3H-radioactivity reached a peak in the serum approximately 20-30 min after injection. Counts were also found in the lens, aqueous and vitreous humors. HPLC using a C18 Bondapak column (37 x 300 mm) indicated that the majority of the 3H-radioactivity in the lens was found in a component of a lower molecular weight than glutathione, but 8.1% of the counts occurred in the peak corresponding to reduced glutathione. Analysis of the unidentified radioactive component revealed a mobility the same as that of a dipeptide. Further analysis suggested this contained the amino acids cysteine and glycine bound in peptide linkage. These results suggest that glutathione may be degraded by the gamma-glutamyl cycle, and the action of transpeptidase produced cysteinylglycine. To confirm these results, similar experiments were undertaken using 35S-glutathione injection, to test whether a differently labelled form would be able to enter the lens. Homogenates prepared from the lens 20 min after 35S-glutathione injection were fractionated by HPLC. The glutathione peak contained 4.5% of the radioactivity in the lens extract. This amount was similar in quantity to the value for 3H-glutathione uptake by the lens. The average of the two values indicated that 6.3% of the total lens label was glutathione. The source of the labelled glutathione taken up by the lens was investigated by determining its concentration in the aqueous and vitreous humors and serum. The dipeptide appeared to be the major radiolabelled form occurring in the serum. This may explain its high level in the lens, as a result of uptake from other sources. Analysis using HPLC revealed that reduced glutathione (GSH) was the predominant chemical species of glutathione in the aqueous humor. In the vitreous humor, oxidized glutathione (GSSG) was the major species. The ratio of GSSG:GSH in the vitreous varied between 2:1 and 4:1. CONCLUSIONS: Over a 4 h period the lens could obtain 12.3% of its total GSH from the injected GSH, using the specific activity of the labelled glutathione to calculate the actual uptake of glutathione by the lens, suggesting a half-time of 16.25 h for replenishing GSH from external sources. The probable route of glutathione entry was by blood plasma and aqueous since the specific activity of the vitreous humor was too low for the vitreous to be a possible source of the lens GSH.

Animals↗
PubMed1999

Modelling cortical cataractogenesis 21: in diabetic rat lenses taurine supplementation partially reduces damage resulting from osmotic compensation leading to osmolyte loss and antioxidant depletion.

The concentration of taurine and the amino acids, glutathione, cysteine, ascorbate and ATP were determined in the lenses of rats made diabetic with streptozotocin. In the clear lenses, prior to vacuole formation after 1 or 2 weeks of diabetes, the increase in concentration of sorbitol and the total decrease of all these osmolytes were not significantly different. The major components of the osmolytes lost were taurine and amino acids, which together accounted for over 75% of the total osmolyte loss. Since glutathione, ascorbate, taurine and cysteine have been reported to have antioxidant activity, it appears that their loss may potentiate damage occurring as a result of free radicals generated by nonenzymic glycation by the Maillard reaction. Amino acids also lost as a result of the osmotic compensation, are estimated to be responsible for almost half of the antioxidant activity lost. To test this hypothesis, normal and streptozotocin diabetic female Wistar rats were given taurine at 0.05% or 0.10% (w/w) in the diet. This treatment resulted in small only marginally significant increases in serum taurine levels. At the end of 6 weeks the rats were examined for weight gain or loss and at the time of killing, blood was collected for measurement of serum glucose. gamma-Crystallin levels were determined in vitreous and aqueous humours using a radioimmunoassay. A lens from each rat was homogenized in 8 m guanidinium chloride for adenosine triphosphate (ATP) analysis. In normal rats, a small amount of gamma-crystallin was found in the vitreous humour, and an even smaller amount in the aqueous humour. Diabetes caused a 4- to 5-fold increase in the vitreous humour and a 4-fold increase in gamma-crystallin in the aqueous humour. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens ATP levels. Addition of taurine to the diet of diabetic animals resulted in a significant decrease of gamma-crystallin leakage into the vitreous but not the aqueous humour. Taurine had no effect on the lens ATP levels. Neither streptozotocin diabetes nor taurine in the diet appeared to affect the weight of the lenses.

Adenosine Triphosphate↗
PubMed1999

Modelling cortical cataractogenesis 22: is in vitro reduction of damage in model diabetic rat cataract by taurine due to its antioxidant activity?

The protective effect of taurine in model in vitro diabetic cataract and the mechanism of this effect were investigated in isolated rat lenses. Isolated rat lenses were incubated in medium 199 in elevated glucose (55.6 m m) with taurine (5 m m). Taurine concentrations in the lenses were determined by amino acid analysis. Accumulative leakage of the intracellular enzyme lactate dehydrogenase (LDH) was used to estimate damage to the lens, as previously reported. In the clear lenses, prior to vacuole formation, after 1 or 2 days of incubation, the taurine and amino acids in lenses decreased progressively in concentration. In lenses incubated with 5 m m taurine, the level of taurine was increased towards that of control lenses. In taurine-treated lenses LDH leakage was significantly decreased, and lens clarity was maintained, similarly to that found previously for vitamin C and lipoic acid. To test whether taurine has similar antioxidant activity, we tested its ability to decrease luminol luminescence generated by (1) superoxide from hypoxanthine/xanthine oxidase and (2) peroxide from diluted glucose/glucose oxidase. For either superoxide or peroxide, the luminescence was decreased to zero, as a function of increasing taurine concentration, at 30 m m, approximately the physiological concentration of taurine in the lens. Spin trapping confirmed that taurine scavenged superoxide. This is consistent with a role for taurine as an important antioxidant protecting the lens against oxidative insults. Amino acids also had antioxidant activity in this assay, and as a group, when all activities were summed, their loss also contributed significantly to the antioxidant loss. Taken in conjunction with Wolff and Crabbe's observation of increased free radical generation by glucose auto-oxidation in diabetes, this suggests a push-pull mechanism for increased oxidative stress in diabetic cataract, involving both increased free radicals and decreased radical scavenging antioxidants.

Amino Acids↗
PubMed1999

Mitochondria of rat lenses: distribution near and at the sutures.

PURPOSE: This study is part of an effort to clarify mitochondrial distribution in the lens in order to better understand lens metabolic function. This study of the rat lens involves: 1) Using confocal microscopy, Rhodamine-123 and Calcium Green fluorescent dyes, to characterise the distribution of mitochondria and calcium in whole rat lenses of different ages in epithelial and superficial cortical fibre cells approaching sutures and 2) Using a scanning laser system to measure the optical quality at the sutures. METHODS: Lenses of rats from age 1 week to 22 months were pre-incubated for 24 hrs in 1.5 ml medium 199 (M199). Those exhibiting damage, as evaluated by protein leakage or visual opacities, were discarded. Lenses were labelled with 50 microg/ml Calcium Green for 45 min and/or 14 microM Rhodamine-123 for 25 min and embedded in 1% agarose in M199 for inverted laser scanning confocal microscopy with a 40 x water immersion lens. The lens optical properties were determined with a scanning laser system. RESULTS: Lens focal length variability significantly increased at the sutures of 13 month-old lenses, the only age investigated. An absence of both mitochondria and calcium was observed at the sutures in rat lenses of all ages. Elongated (up to 108 mm) mitochondria were present in superficial cortical fibre cells approaching the sutures of 16 month-old lenses. Calcium Green fluorescent staining was seen closer to the border of the suture, where mitochondria were absent. Along the axis, 1 week-old lenses showed a mitochondria free zone (MFZ) starting 177 microm below the lens surface, whereas in 22 month-old lenses the MFZ started only 29 microm below the surface. In the equatorial fibre cells, mitochondria were seen to a depth of 220 microm. CONCLUSIONS: Optical quality near and at the suture decreased in 13 month-old lenses despite the reduction in light scattering that should be associated with absence of mitochondria at the sutures. This suggests that mitochondrial loss in superficial cortical fibre cells may originate at the sutures and may compensate for loss of optical quality at the sutures.

Animals↗
PubMed1999

Uptake of vitamin E succinate by the skin, conversion to free vitamin E, and transport to internal organs.

The percent solubility at 34 degrees C (skin temperature) of radioactive tocopherol succinate was determined for a number of edible oils, and a semisynthetic oil, Myritol 318 (Henkel, Kankakee, IL, a medium chain triglyceride prepared from fractionated coconut oil). Its solubility in Myritol 318 was approximately 50% better than any of the other oils. 14C-tocopherol succinate was diluted (1) into pure Myritol 318, a cosmetic base or (2) 50% tocopherol succinate in Myritol 318. These preparations were applied topically to a 2 cm diameter circle of the back saddle skin of a hairless mouse (strain skh-1). After 24 hr, up to 65% of the label was absorbed by the skin and was also found in skin removed from areas of the back other than the application area, and internal organs such as liver and heart. Up to 6% was hydrolysed to free tocopherol. Topical treatment may be an alternative to oral administration in gastrointestinal malabsorption diseases.

Administration, Cutaneous↗
PubMed1999

Is ethanol an important antioxidant in alcoholic beverages associated with risk reduction of cataract and atherosclerosis?

It has been reported in the epidemiological literature that cataract, stroke, and atherosclerosis risk is reduced by 50% in people consuming one alcoholic drink per day. Peroxide has been implicated as a causative agent in cataractogenesis, and LDL oxidation appears to play a role in atherosclerosis. The antioxidant activity of alcohol was measured by: (i) use of a luminescent assay developed in our laboratory, confirmed as appropriate; (ii) electron spin resonance (ESR) spin-trapping; and (iii) copper-catalysed oxidation of LDL and VLDL from hamsters fed 6% ethanol in their drinking water. Ethanol reduced the luminescent counts/min from peroxide and superoxide. It significantly reduced the spin-trapped signal of hydroxyl radical, but not the superoxide signal. Other alcohols also showed large reductions in counts from hydrogen peroxide. Plasma from hamsters fed 6% ethanol had lower lipid peroxides and the oxidizability of LDL and VLDL was significantly reduced compared to controls. These data provide a possible explanation for the effect of beverages containing ethanol in the reduction of cataract and atherosclerosis risk observed in human population studies.

Alcohol Drinking↗
PubMed1998

Modelling cortical cataractogenesis. XXIX. Calpain proteolysis of lens fodrin in cataract.

The relation between cataract and calpain proteolysis of lens fodrin was studied in two systems: elevated glucose (55.6 mM, diabetic model), and cytochalasin D (CD, 10(-2) mM, actin depolymerization-induced opacity model). Glucose treatment (48 h) caused a visible opaque layer and enzyme leakage, with a concomitant accumulation of ([Ca2+]i) around the lens equatorial cortex. CD caused both earlier and greater opacity and enzyme leakage than glucose. Lens fodrin digestion occurred in parallel with the timing and extent of calcium elevation. A calpain inhibitor peptide (CIP, 10(-2) mM) reduced the proteolysis of fodrin, opacity, and enzyme leakage in glucose-treated lenses but only partially retarded them in CD-treated lenses. These results suggest a mechanism in which calpain proteolysis of fodrin is a critical event in lens damage during opacification of cortical cataract.

Amino Acid Sequence↗
PubMed1998

Modelling cortical cataractogenesis XX. In vitro effect of alpha-lipoic acid on glutathione concentrations in lens in model diabetic cataractogenesis.

In previous studies stereospecific protection against lens opacity was consistent with specific reduction of R-alpha-lipoic acid(R-alpha-LA) in mitochondria of the vulnerable cells at the lens equator where the first globular degeneration is seen in glucose cataract. In this study two further possible explanations of this effect were investigated: (1) increased glucose uptake by the lens, leading to increased glycolysis and release of lactate into the incubation medium and/or (2) maintenance of glutathione levels by the R-alpha-LA. The data did not support 1, but was consistent with 2, after 24 hr incubation. The concentrations of glutathione in normal lenses or lenses incubated with R- or racemic alpha-LA were not significantly different, but the concentration of glutathione in lenses incubated with S-alpha-LA was significantly lower than the R-alpha-LA-incubated lenses.

Animals↗
PubMed1997

Cysteine and ascorbate loss in the diabetic rat lens prior to hydration changes.

PURPOSE: Glutathione (GSH) loss precedes vacuole formation in the diabetic rat lens, but the cause of this loss is not known. Cysteine availability is a rate limiting factor to glutathione biosynthesis in rat and human lenses but its concentration is not known; therefore free cysteine was measured prior to lens hydration in the diabetic rat lens. GSH can regenerate ascorbate from dehydroascorbate within the lens and potentially modulate the ascorbate pool; therefore ascorbate loss is also a possibility that has not been examined previously. METHODS: Diabetes was induced in Wistar rats to provide a slowly progressing model of cortical cataract. Age-matched control rats were injected with buffer vehicle only. Lens condition was monitored by binocular slit-lamp microscope after pupil dilation. Lens cysteine and glutathione were measured in the same lens, while ascorbate and total ascorbate (ascorbate + dehydroascorbate) of the contralateral lens were quantified by high performance liquid chromatography electrochemical detection. The 1- and 2-week periods of diabetes were chosen as they both preceded lens hydration changes and Na+/K+ changes, to avoid leakage due to ruptured cell membranes. RESULTS: Lens weights were not significantly different compared to controls at either the 1- or 2-week periods, and lenses were completely free of initial vacuole formation. Lens GSH concentration was diminished by 72% compared with controls after 1 week of diabetes and 74% after 2 weeks of diabetes. Lens free cysteine was decreased by 62% and 78% compared with controls after 1 and 2 weeks of diabetes, respectively. Total lens ascorbate concentration was decreased by 34% after 1 week of diabetes and 48% after 2 weeks of diabetes. Dehydroascorbate levels represented less than 10% of the total lens ascorbate pool in all experimental groups. GSH and ascorbate concentration were correlated after 1 week of diabetes (p < 0.005) and after 2 weeks of diabetes (p < 0.001). GSH and cysteine concentration were also correlated after 1 week of diabetes (p < 0.001) and after 2 weeks of diabetes (p < 0.001). CONCLUSIONS: Decreased free cysteine, in the diabetic rat lens, precedes hydration changes and vacuole formation, contributing to decreased glutathione content. While cysteine was not abundant in the lens, its concentration is greater than previously supposed. The lens ascorbate pool was also diminished prior to lens hydration.

Animals↗
PubMed1997

Endogenous superoxide-like species and antioxidant activity in ocular tissues detected by luminol luminescence.

A new luminescent method was used to detect the reactive oxygen species in aqueous and vitreous humors and in homogenates of the lens and retina of laboratory rats. Superoxide-like activity per microgram protein increased in all tissues with weight of the rat, a good indicator of animal age. Superoxide dismutase, centrophenoxine, soluble vitamin E (D-alpha-Locopherol (polyethlyene glycol 1000) succinate, and N'-diphenyl-p-phenylenediamine (DPPD) reduced the luminescence. Catalase had no effect. These results are consistent with the detected species being superoxide-like.

Age Factors↗
PubMed1997

Antioxidants and cataract: (cataract induction in space environment and application to terrestrial aging cataract).

The effect of several antioxidants and cysteine-elevating precursor drugs (prodrugs) was tested on lens damage occurring after in vitro exposure to low levels of 60Co-gamma-irradiation, to simulate in vitro the exposure to radiation in vivo of (1) astronauts (2) jet crews (3) military radiation accident personnel. Tocopherol (100 microM), ascorbic acid (1 mM), R-alpha-lipoic acid (1 mM), and taurine (0.5 mM) protected against radiation-associated protein leakage. MTCA and ribocysteine protected lenses against opacification, LDH and protein leakage, indicating that antioxidants and prodrugs of cysteine appear to offer protection against lens damage caused by low level radiation.

Animals↗
PubMed1996

Modelling cortical cataractogenesis. XVIII. In vitro diabetic cataract reduction by venoruton. A flavonoid which prevents lens opacification.

The effect of a novel flavonoid, venoruton (a mixture of mono-, di-, tri- and tetrahydroxyethylrutosides) has been investigated in healthy rat lenses and compared with diabetic cataract modelled in vitro. One mM venoruton was added to medium simulating healthy and diabetic conditions for the incubated lenses; damage was followed by either stereoscopic photography of the lenses under a Cooperative Cataract Research Group operating microscope or with our recently developed method: the leakage of lactate dehydrogenase (LDH) into the lens culture media. The increased LDH activity in the medium and observable development of the opacity were correlated with cell damage, which has been found to be associated with globular degeneration and cataract formation. The extent of opacification and LDH release is reduced if 1 mM venoruton is included in the medium. The protective effect may be related to antioxidant activity against reactive oxygen species: decreased luminol luminescence was shown after venoruton addition to either superoxide-generating hypoxanthine plus xanthine oxidase, or hydrogen peroxide.

Animals↗
PubMed1995

Vitamin C reduces cytochalasin D cataractogenesis.

The effect of cytochalasin D (CD), an actin monomer-stabilizer, has been studied on cataract development in rat lenses. Cataractogenesis was induced by incubating the rat lenses in medium 199 (M199) containing 10(-5) M CD; by the end of 24 h, lenses first developed a visible opacity. The increased lactate dehydrogenase (LDH) activity in the culture medium, leakage of lens cytosolic proteins into the culture medium and observable development of opacity through a dissection microscope were correlated with cell damage associated with cataract formation. Non-denaturing polyacrylamide gel electrophoresis was used to separate three lens LDH isoenzymes. The effect of 1 mM vitamin C (VC) in reducing LDH leakage was also examined. The protective effect of VC on CD-initiated cataractous lenses is significant. This suggest that a portion of the opacity and lens damage may involve oxidative damage to the membrane-cytoskeleton complex which is started by CD, but partially prevented by VC

Animals↗
PubMed1995

Modelling cortical cataractogenesis. 16. Leakage of lactate dehydrogenase: a new method for following cataract development in cultured lenses.

The in vitro effect of glucose on cultured rat lenses was correlated with (i) the time-dependent leakage of lactate dehydrogenase (LDH) into defined medium; (ii) the appearance of the lens under the dissection microscope; and (iii) the leakage of lens cytosolic proteins. A protective effect of 1 mM Vitamin C (VC) was also found: the extent of opacification, LDH and gamma-crystallin release were reduced if 1 mM VC was included in the medium. Using the above parameters provides a new and more rapid technique to follow lens opacification in vitro.

Animals↗
PubMed1995

Modelling cortical cataractogenesis 17: in vitro effect of a-lipoic acid on glucose-induced lens membrane damage, a model of diabetic cataractogenesis.

The effect of R, S, and racemic forms of a-lipoic acid was tested on the formation of opacity in normal rat lenses incubated with 55.6 mM glucose, as a model for in vivo diabetic cataractogenesis. Control lenses, incubated 8 days with 5.56 mM glucose, did not develop opacities. Formation of lens opacities in vitro was correlated with lactate dehydrogenase (LDH) leakage into the incubation medium. Opacity formation and LDH leakage, resulting from incubation in medium containing 55.6 mM glucose to model diabetes, were both suppressed by the addition of 1 mM R-lipoic acid. Addition of 1 mM racemic lipoic acid reduces these damaging effects to the lens by one-half, while S-lipoic acid potentiated LDH leakage, consistent with the hypothesis that R-lipoic acid is the active form. Although HPLC analysis demonstrated that both stereoisomers of lipoic acid were reduced to dihydrolipoate at comparable rates by the intact lens, the mitochondrial lipoamide dehydrogenase system is highly specific for reduction of exogenous R-lipoic to dihydrolipoic acid. Therefore, stereospecific protection against this opacity is consistent with specific reduction of R-lipoic acid in mitochondria of the vulnerable cells at the lens equator where the first globular degeneration is seen in glucose cataract.

Animals↗
PubMed1994

High-performance liquid chromatography-electrochemical detection of antioxidants in vertebrate lens: glutathione, tocopherol, and ascorbate.

The HPLC-EC method has good specificity for the analysis of glutathione, tocopherol, and ascorbate. The same HPLC system can be used for all three analysis with changes of mobile phase and the electrode cell to match the procedure required. The same C18 reversed-phase column has been used with a refillable guard column for 3 years with no noticeable loss of resolving power. The main advantage of the glutathione procedure was the ability to monitor both GSH and GSSG, which allowed us to confirm that loss of GSH in the diabetic rat lens does not result in the appearance of GSSG. The main benefit of the tocopherol procedure was the ability to measure the tocopherol content of a single rat lens. Our previous experience with UV or fluorescence detection showed those methods to be not sensitive enough for a single lens determination. The mammalian lens has the lowest tocopherol content of the tissues of the eye, 10 to 40 times less than most body tissues as measured by gas chromatography-mass spectrometry (GC-MS). The better sensitivity of electrochemical detection has allowed for a single lens determination, keeping the number of experimental animals to a minimum. An advantage of the ASC analysis procedure was the extra specificity imparted by both the chromatography and the detector as well as the ability to estimate the total ascorbate (ASC plus DHAA) and DHAA content. Other reducing agents such as GSH and uric acid can interfere in colorimetric methods that rely on the reducing action of ASC. The very high GSH content of the mammalian lens was a concern when choosing a procedure. GSH levels exceeding 10 times the level of lens samples were found to yield no response using the HPLC-EC procedure for ASC. The only disadvantage with electrochemical detection was that the electrode response could drift with time, requiring more frequent calibration with standards. We continue to utilize these methods to examine the prevacuole loss of ASC and GSH in the diabetic rat lens model of cataract.

Animals↗