Lens redox fluorometry: Pyridine nucleotide fluorescence and analysis of diabetic lens

Tsubota, Kazuo; Krauss, Joel M.; Kenyon, Kenneth R.; Laing, Ronald A.; Miglior, Stefano; Cheng, Hong

doi:10.1016/0014-4835(89)90043-2

Cited by 13 publications

(6 citation statements)

References 26 publications

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“…Finally, the increases in polyol (sorbitol) and fructose levels and in the UP ratio as well as the reduced myo-inositol levels observed in cells cultured in 30 mM glucose indicate that changes in polyol pathway activity, myo-inositol metabolism, and cytosolic redox state were produced and maintained for the entire 4-week period under these experimental conditions, in keeping with previous reports concerning mesangial and other cells and tissues (62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74).…”

Section: Discussionsupporting

confidence: 89%

Mechanisms of Glucose-Enhanced Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells

et al. 1994

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In view of the importance of mesangial extracellular matrix (ECM) accumulation in the pathogenesis of diabetic glomerulosclerosis, we investigated 1) the effects of high glucose on ECM production by rat glomerular mesangial cells in culture (study A) and 2) the mechanisms underlying these effects, particularly the role of high sugar levels irrespective of intracellular metabolism (study B1) and of excess glucose disposal via the polyol pathway and associated biochemical alterations (study B2). Cells were cultured for 4 weeks, through six to eight passages, under the experimental conditions indicated below and, at each passage, the levels of fibronectin (FN), laminin (LAM), and collagen types I (C-I), III (C-III), IV (C-IV), and VI (C-VI) in media and cell extracts were quantified by an enzyme immunoassay. In study A, medium and cell content of matrix were assessed, together with [3H]leucine and [3H]thymidine incorporation into monolayers, polyol, fructose, and myo-inositol levels and the cytosolic redox state, in cells grown in high (30 mM) D-glucose or iso-osmolar mannitol versus cells cultured in normal (5.5 mM) D-glucose. FN, LAM, C-IV, and C-VI accumulation, but not C-I and C-III accumulation, was increased by 30 mM glucose, but not by iso-osmolar mannitol, when compared with 5.5 mM glucose, starting at week 2 and, except for C-VI, persisting throughout the remaining 2 weeks, whereas no change was observed in the measured indexes of total protein synthesis and DNA synthesis/cell proliferation. At any time point, polyol levels were increased, whereas myo-inositol was reduced by high glucose; in cells grown under elevated glucose concentrations, the lactate/pyruvate (L/P) ratio, an index of the cytosolic redox state, progressively increased. In study B1, the effects of high D-glucose were compared with those of iso-osmolar concentrations of sugars that are partly or not metabolized but are capable of inducing nonenzymatic glycosylation, such as D-galactose and L-glucose, and of mannitol, which does not enter the cell. Both D-galactose and L-glucose, but not mannitol, partly mimicked D-glucose-induced ECM overproduction. Although D-galactose is metabolized via the polyol pathway and alters the cytosolic redox state, ECM changes induced by high galactose were not prevented by the use of an aldose reductase inhibitor (ARI), Alcon 1576 (14 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Discussionsupporting

confidence: 89%

Mechanisms of Glucose-Enhanced Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells

et al. 1994

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“…Under very high magnification, in this experiment, fluorescence spectra are also detectable for 446 and 470 nm excitation and have maxima at 514 and 528 nm, respectively. As the fluorescence maximum of NADH is known at 450 nm from the literature (Cordeiro et al, 1995;Kobayashi et al, 2002;Tsubota et al, 1989), the fluorescence maxima, detected here, probably originate from pollution. In contrast to measurements at the fundus, the autofluorescence of NADH is detectable from the anterior part of the eye, e.g., from the lens.…”

Section: Materials and Methods Excitation And Emission Spectra Of Expsupporting

confidence: 61%

Towards metabolic mapping of the human retina

Schweitzer

Schenke

Hammer

et al. 2007

Microscopy Res & Technique

206

240

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Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40 degrees fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 +/- 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 approximately 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus.

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“…1A and 1C, and in vitro in Figs. 2 and 3, has been reported previously for the rabbit lens (Pitts, 1978; Tsubota et al, 1989), and cultured rabbit lens epithelial cells (Atherton et al, 1999). Based on the in vitro experiment described in the Results, the fluorescence is apparently due to absorption of 365 nm light by NADPH and NADH.…”

Section: Discussionsupporting

confidence: 63%

“…Based on the in vitro experiment described in the Results, the fluorescence is apparently due to absorption of 365 nm light by NADPH and NADH. Other researchers have used redox fluorometry to show that 99% of the pyridine nucleotide blue fluorescence arising from the rabbit lens is due to absorption by NADH (Tsubota et al, 1989). The rabbit lens contains unusually high levels of NADH, four and eight times higher than lenses of the rat and human, respectively (Giblin and Reddy, 1980), due in part to the presence of NADH bound to λ-crystallin (Bando et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

A Class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo

Giblin¹,

Lin²,

Simpanya³

et al. 2012

Experimental Eye Research

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It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm2 on the cornea) for 1 hour using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 hour. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear λ-crystallin after the in vivo UVA exposure. It is concluded that UVA-induced loss of free NADH (which fluoresces blue) may have allowed the natural yellow fluorescence of λ-crystallin and other proteins in the lens nucleus to become visible. Increased fluorescence exhibited by UVA-exposed λ-crystallin may have been the result of a UVA-induced change in the conformation of the protein occurring during the initial UVA-exposure in vivo. The results demonstrate the greater susceptibility of the lens nucleus to UVA-induced stress, and may relate to the formation of human nuclear cataract. The senofilcon A contact lens was shown to be beneficial in protecting the rabbit lens against effects of UVA light, including changes in fluorescence, increased yellowing and loss of pyridine nucleotides.

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Lens redox fluorometry: Pyridine nucleotide fluorescence and analysis of diabetic lens

Cited by 13 publications

References 26 publications

Mechanisms of Glucose-Enhanced Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells

Mechanisms of Glucose-Enhanced Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells

Towards metabolic mapping of the human retina

A Class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo

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