M. Christine McGahan

The determination of platinum in biological materials by electrothermal atomic absorption spectroscopy

Abstract Methods for determination of platinum in body tissues and fluids by electrothermal atomic absorption spectroscopy are described. Serum and urine could be analyzed without pretreatment or dilution. Wet and dry ashing techniques for tissue digestion were compared. Dry ashing tissues in a furnace resulted in significant and unexplained losses of analyte, whereas there was complete recovery of platinum added to the tissues when the tissues were wet ashed. The wet ashing technique is fast and convenient and requires minimal sample treatment.

Alpha lipoic acid changes iron uptake and storage in lens epithelial cells

Alpha lipoic acid (LA) is a cofactor in mitochondrial dehydrogenase complexes. Previous studies have shown that when administered exogenously LA has antioxidant properties, which include free radical scavenging, metal chelation and regeneration of other antioxidants. The cells convert LA into dihydroplipoic acid (DHLA), which in the presence of iron can act as a prooxidant. In vitro DHLA reduces Fe(+3) to Fe(+2) and removes iron from ferritin, increasing the risk of Fe catalyzed free radical formation. In the present study we examined the in vivo effects of lipoic acid treatment on Fe metabolism in cultured lens epithelial cells, and found that LA decreases Fe uptake from transferrin, increases Fe deposition into ferritin and increases the concentration of this protein. When administered together with ascorbic acid, lipoic acid changes the characteristic heavy to light chain ratio of ferritin makeup. The decreased Fe uptake and increased storage diminishes the size of the cytosolic highly reactive Fe pool (LIP). These changes are associated with increased cell resistance to H(2)O(2) challenge. Therefore, LA may reduce the risk of Fe induced oxidative damage and also might be useful as a treatment of Fe overload.

The lens influences aqueous humor levels of transforming growth factor-β2

Abstract · Background: Transforming growth factor-beta 2 (TGF-β2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid (IOF) levels of TGF-β2 are quite high. Although the sources of ocular TGF-β are not completely defined, the retinal pigment epithelium, the epithelium of the ciliary body and trabecular meshwork cells all secrete it. In this study we utilized canine lens and rabbit ciliary pigmented epithelial cell cultures to quantitate the in vitro secretion of TGF-β2. In addition, the effects of aphakia or the presence of cataractous lenses on IOF TGF-β2 levels were determined. · Methods: Lens and ciliary body epithelial cell culture supernatants and aqueous humors were assayed for total TGF-β2 levels by ELISA and bioassay. · Results: TGF-β2 accumulated in the media bathing lens epithelial cell cultures (0.7 ± 0.03 ng/ml at day 2) and ciliary pigmented epithelial cell cultures (0.8 ± 0.06 ng/ml at day 2) in a time-dependent manner. Surprisingly, aqueous humor from aphakic rabbit eyes contained significantly higher levels of TGF-β2 than their contralateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-β2 than normal eyes. · Conclusions: These results suggest that the lens secretes TGF-β2 and that the presence and status of the lens may influence IOF TGF-β2 levels.

Identification of a mechanism by which lens epithelial cells limit accumulation of overexpressed ferritin H-chain

The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H- or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Forty-eight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H- and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of H-ferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.

Effect of surgical technique on in vitro posterior capsule opacification

Purpose: To compare the effect of different cataract extraction surgical techniques on residual lens epithelial cell (LEC) density and cell regrowth rates using an in vitro model of posterior capsule opacification (PCO). Setting: Comparative Ophthalmology Research Laboratories, North Carolina State University, Raleigh, North Carolina, USA. Methods: Lens capsule explants were prepared from freshly enucleated canine globes after extracapsular cataract extraction (ECCE), phacoemulsification, or phacoemulsification followed by capsule vacuuming. Initial cell density on the capsule and cell proliferation were determined by phase contrast microscopy. The effects of the surgical technique on time to confluent growth of the cells across the posterior lens capsule were determined. Results: Residual cell density on the remaining anterior capsule immediately after lens removal was 31.6% ± 19.3%, 16.1% ± 8.9%, and 7.7% ± 5.7% in the ECCE, phacoemulsification, and phacoemulsification/capsule‐vacuuming groups, respectively. Time to confluence (range 5.0 to 6.3 days) was not significantly different among the 3 groups when the lens capsules were cultured in serum‐supplemented media. The confluence rate was significantly longer (by approximately 5 to 7 days) in the phacoemulsification/capsule‐vacuuming group than in the other 2 groups when the capsules were cultured in serum‐free media. Conclusions: Phacoemulsification with and without anterior and equatorial capsular vacuuming led to less initial LEC density in the capsular bag than ECCE. However, because cell proliferation rates among the 3 groups were only marginally affected, near 100% removal of LEC at the time of cataract extraction may be necessary to prevent PCO.

Ascorbic acid levels in aqueous and vitreous humors of the rabbit: Effects of inflammation and ceruloplasmin*

A HPLC method for determination of ascorbic acid and dehydroascorbic acid in plasma and aqueous and vitreous humors of rabbits is described. Values for total ascorbic acid concentration found in this study are in agreement with those of previous investigators. Endotoxin-induced ocular inflammation caused a decrease in the concentration of ascorbic acid in the aqueous humor and an increase in the vitreous humor. The additional lack of correlation between levels of ascorbic acid in normal aqueous and vitreous humors from the same uninflamed eye indicates that the aqueous humor is not the source of vitreal ascorbic acid. The copper concentration of the aqueous humor is increased during ocular inflammation, most likely due to the influx of ceruloplasmin from plasma when the blood-aqueous barrier is disrupted. Ceruloplasmin caused a decrease in the amount of ascorbic acid in the aqueous humor in vitro and the vitreous humor in vivo. The presence of ceruloplasmin in the aqueous humor during inflammation thus may contribute to the decreased concentration of ascorbic acid in this fluid.

Iron Regulates L-Cystine Uptake and Glutathione Levels in Lens Epithelial and Retinal Pigment Epithelial Cells by Its Effect on Cytosolic Aconitase

PURPOSE The authors previously published the novel finding that iron regulates L-glutamate synthesis and accumulation in the cell-conditioned medium (CCM) by increasing cytosolic aconitase activity in cultured lens epithelial cells (LECs), retinal pigment epithelial (RPE) cells, and neurons. The present study was designed to determine whether iron-induced L-glutamate accumulation in the CCM regulates L-cystine uptake and glutathione (GSH) levels through the aconitase pathway in LECs and RPE cells. METHODS The presence of xCT, the light chain of X(c)(-), a glutamate/cystine antiporter, was analyzed by RT-PCR, immunoblotting, and immunocytochemistry. Uptake of L-[(35)S]cystine and L-[(3)H]glutamate was measured in the presence or absence of transporter inhibitors. L-cystine uptake and intracellular GSH concentration were measured in the presence or absence of iron-saturated transferrin, the iron chelator dipyridyl (DP), or oxalomalic acid (OMA), an aconitase inhibitor. RESULTS LECs and RPE cells express xCT, as evidenced by RT-PCR analysis and immunoblotting. xCT was localized by immunocytochemistry. The authors found that the iron-induced increase in L-glutamate availability increased L-cystine uptake, with subsequent increases in GSH levels. In addition, L-glutamate production, L-cystine uptake, and GSH concentration were inhibited by OMA and DP, indicating a central role for iron-regulated aconitase activity in GSH synthesis in LECs and RPE cells. CONCLUSIONS These results demonstrate for the first time that iron regulates L-cystine uptake and the downstream production of GSH in two mammalian cell types. It is possible that the increase in intracellular antioxidant concentration induced by iron serves as a protective mechanism against the well-established capacity of iron to induce oxidative damage.

Determination of copper concentration in blood plasma and in ocular and cerebrospinal fluids using graphite furnace atomic absorption spectroscopy

Published values for the concentration of Cu in cerebrospinal and intraocular fluids cover a very wide range (0.016 to 1.0 microgram/ml) and include values which are several times higher than those which would be consistent with normal physiology. An atomic absorption spectrophotometer equipped with a graphite furnace was used to measure the Cu concentration in these fluids and in blood plasma of toads, rabbits, and cats. Under standard conditions, these fluids yielded high background absorbance and only fractional recovery of added Cu. Parameters were therefore established which eliminated both the high background and the matrix interference and allowed the determination of Cu in 10-microliters aliquots of diluted blood plasma and undiluted cerebrospinal and ocular fluid samples. Under these conditions the Cu measured in the ocular (0.011 to 0.032 microgram/ml) and cerebrospinal fluids (0.033 to 0.050 microgram/ml) of these three species was lower than most previously reported values and only a small fraction (1-3%) of the concentration of Cu in the plasma of the same animals (0.85 to 1.22 micrograms/ml).

A micromethod for the determination of iron and total iron-binding capacity in intraocular fluids and plasma using electrothermal atomic absorption spectroscopy.

A new micromethod (requiring only 30 microliters of sample) for the determination of iron (Fe) concentration and total iron-binding capacity (TIBC) in intraocular fluids (IOFs; aqueous and vitreous humors) and plasma has been developed using electrothermal atomic absorption spectrophotometry. Values for Fe concentration in the IOFs were similar between species and were generally higher in the vitreous (0.031-0.060 mg/liter) than aqueous (0.014-0.038 mg/liter) humors obtained from the same eye. Plasma Fe concentration and TIBC fell within established limits for all species studied. The iron binding capacity of the aqueous and vitreous humors from the same species is similar. While the TIBC of the aqueous humor is saturated to the same extent as plasma, vitreous TIBC is more highly saturated in all species. Using this method considerably less species variation in IOF Fe concentration was found as compared to previous studies. Some possible methodological reasons for this difference are discussed.

Endotoxin-induced ocular inflammation increases prostaglandin E2 synthesis by rabbit lens

Twenty-four hours after the intravitreal injection of 0.1-100 ng of Escherichia coli endotoxin into one eye of the New Zealand white rabbit, lenses from the inflamed eyes synthesized significantly more prostaglandin E2 (PGE2) than their contralateral, control lenses at all doses of endotoxin greater than 0.1 ng. PGE2 elevations were also seen in the aqueous and vitreous humors from inflamed eyes. Lenses did not synthesize 6-keto-PGF1alpha (a stable metabolite of PGI2). Incubation of untreated lenses with 1 microgram ml-1 of endotoxin for 24 h did not increase PGE2 production. These results indicate that rabbit lens can synthesize PGE2, that this synthesis is significantly increased 24 hr after the intravitreal infusion of E. coli endotoxin, and that this increased PGE2 synthesis is most likely not due to a direct action of endotoxin on the lens.