Optical characterization of melanin.

The optical properties of melanin have been characterized for a number of laser wavelengths in the visible region. The index of refraction of melanin is measured by the conventional method of minimum deviation using a hollow quartz prism at these wavelengths. The inverse adding doubling method based on the diffusion approximation and radiative transport theory have been employed to determine the absorption, scattering, and scattering anisotropy coefficients of melanin from the measurements of diffuse transmission, diffuse reflection and collimated transmission using double integrating spheres. The results obtained by the use of inverse adding doubling method have been compared to the Monte Carlo simulation technique.

Erbium: YAG versus holmium:YAG lithotripsy.

PURPOSE: We test the hypothesis that erbium:YAG (Er:YAG) lithotripsy is more efficient than holmium:YAG (Ho:YAG) lithotripsy. MATERIALS AND METHODS: Human calculi composed of greater than 97% calcium oxalate monohydrate and cystine were studied. Calculi were irradiated in water using Er:YAG or Ho:YAG lasers. Er:YAG lithotripsy was done with a 425 microm sapphire optical fiber at a pulse energy of 50 mJ at 10 Hz. Ho:YAG lithotripsy was performed with a 365 microm low hydroxy optical fiber at a pulse energy of 500 mJ at 10 Hz or a 425 microm sapphire optical fiber at a pulse energy of 50 mJ at 10 Hz. Fragmentation was defined as the initial stone mass minus the final dominant fragment mass and normalized for incident laser fluence (energy per unit area of fiber tip). RESULTS: Mean fragmentation plus or minus standard deviation for calcium oxalate monohydrate was 38 +/- 27 mg for Er:YAG and 22 +/- 6 for Ho:YAG (low hydroxy silica fiber) versus 5 +/- 1 for Ho:YAG (sapphire fiber, p = 0.001). When fragmentation was normalized for incident laser fluence given different optical fiber sizes, mean fragmentation efficiency was 53.6 +/- 38.7 g-microm2/J for Er:YAG lithotripsy compared with 22.6 +/- 6.4 for Ho:YAG (low hydroxy silica fiber) lithotripsy (p = 0.04). Mean cystine fragmentation was 15 +/- 3 mg for Er:YAG versus 9 +/- 1 for Ho:YAG (sapphire fiber, p = 0.0005). CONCLUSIONS: Er:YAG lithotripsy is more efficient than Ho:YAG lithotripsy.

Laser lithotripsy and cyanide.

BACKGROUND AND PURPOSE: Holmium:YAG lithotripsy of uric acid calculi produces cyanide. The laser and stone parameters required to produce cyanide are poorly defined. In this study, we tested the hypotheses that cyanide production: (1) varies with holmium:YAG power settings; (2) varies among holmium:YAG, pulsed-dye, and alexandrite lasers; and (3) occurs during holmium:YAG lithotripsy of all purine calculi. MATERIALS AND METHODS: Holmium:YAG lithotripsy of uric acid calculi was done using various optical fiber diameters (272-940 microm) and pulse energies (0.5-1.5 J) for constant irradiation (0.25 kJ). Fragmentation and cyanide were quantified. Cyanide values were divided by fragmentation values, and fragment sizes were characterized. To test the second hypothesis, uric acid calculi were irradiated with Ho:YAG, pulsed-dye, and alexandrite lasers. Fragmentation and cyanide were measured, and cyanide per fragmentation was calculated. Fragment sizes were characterized. Finally, Ho:YAG lithotripsy (0.25 kJ) of purine and nonpurine calculi was done, and cyanide production was measured. RESULTS: Fragmentation increased as pulse energy increased for the 550- and 940-microm optical fibers (P < 0.05). Cyanide increased as pulse energy increased for all optical fibers (P < 0.002). Cyanide per fragmentation increased as pulse energy increased for the 272-microm optical fiber (P = 0.03). Fragment size increased as pulse energy increased for the 272-microm, 550-microm, and 940-microm optical fibers (P < 0.001). The mean cyanide production from 0.25 kJ of optical energy was Ho:YAG laser 106 microg, pulsed-dye 55 microm, and alexandrite 1 microg (P < 0.001). The mean cyanide normalized for fragmentation (microg/mg) was 1.18, 0.85, and 0.02, respectively (P < 0.001). The mean fragment size was 0.6, 1.1, and 1.9 mm, respectively (P < 0.001). After 0.25 kJ, the mean amount of cyanide produced was monosodium urate stones 85 microg, uric acid 78 microg, xanthine 17 microg, ammonium acid urate 16 microg, calcium phosphate 8 microg, cystine 7 microg, and struvite 4 microg (P < 0.001). CONCLUSIONS: Cyanide production varies with Ho:YAG pulse energy. To minimize cyanide and fragment size, Ho:YAG lasertripsy is best done at a pulse energy < or = 1.0 J. Cyanide production from laser lithotripsy of uric acid calculi varies among Ho:YAG, pulsed-dye, and alexandrite lasers and is related to pulse duration. Cyanide is produced by Ho:YAG lasertripsy of all purine calculi.

Holmium: YAG lithotripsy: optimal power settings.

PURPOSE: We tested the hypothesis that holmium:YAG laser lithotripsy speed is best maximized by using low pulse energy at high pulse frequency. MATERIALS AND METHODS: To demonstrate that optical fiber damage increases with pulse energy and irradiation, the 365-microm optical fiber irradiated calcium hydrogen phosphate dihydrate (CHPD), calcium oxalate monohydrate (COM), cystine, magnesium ammonium phosphate hexahydrate (MAPH), and uric acid calculi at pulse energies of 0.5 to 2.0 J. Optical energy output was measured with an energy detector after 10 J to 200 J of total energy. To demonstrate that lithotripsy efficiency varies with power, fragmentation was measured at constant power settings at total energies of 200 J and 1 kJ with the 365-microm optical fiber. Fragmentation was measured for the 272-microm optical fiber at pulse energies of 0.5 J to 1.5 J at 10 Hz. To demonstrate that low pulse energy produces smaller fragments than high pulse energy, fragment size was characterized for COM and uric acid calculi after 0.25 kJ of irradiation using the 272-microm to 940-microm optical fibers at 0.5 J to 1.5 J. RESULTS: Damage to the 365-microm optical fiber was greatest for irradiation of CHPD, followed by MAPH, and COM (P<0.001). There was no significant optical fiber damage after cystine and uric acid lithotripsy. For the 365-microm optical fiber and CHPD, fragmentation after 200 J was greatest for pulse energies < or =1.0 J (P< 0.001). For other compositions, fragmentation was not statistically different among the power settings for constant irradiation. No significant difference was noted in fragmentation for any composition at different pulse energies (1.0 v. 2.0 J) for 1-kJ irradiation. However, for all compositions, the calculated lithotripsy speed was greatest at high power settings (P<0.001). For the 272-microm optical fiber, CHPD fragmentation was greatest for the 1.0-J pulse energy. The mean fragment size and relative quantity of fragments > or =2 mm both increased as pulse energy increased. CONCLUSIONS: Optical fiber degradation varies with stone composition, irradiation, and pulse energy. Holmium:YAG lithotripsy speed is maximized with higher power (either increased pulse energy or higher pulse frequency). Because low pulse energy may be safer and yields smaller fragments than high pulse energy, holmium:YAG lithotripsy speed is best increased by using pulse energies < or =1.0 J at a high repetition rate.

Proteus mirabilis viability after lithotripsy of struvite calculi.

PURPOSE: We tested the hypotheses that Proteus mirabilis viability of struvite calculi differs after exposure to different lithotripsy modalities and that the photothermal mechanism of holmium:YAG lithotripsy is antibacterial. MATERIALS AND METHODS: Human calculi of known struvite composition (greater than 90% magnesium ammonium phosphate hexohydrate) were incubated with P. mirabilis. Calculi were randomly distributed and fragmented with no lithotripsy (controls), or shock wave, intracorporeal ultrasonic, electrohydraulic, pneumatic, holmium:YAG or pulsed dye laser lithotripsy. After lithotripsy fragments were sonicated and specimens were serially plated for 48 hours at 38C. Bacterial counts and the rate of bacterial sterilization were compared. RESULTS: Median bacterial counts (colony-forming units per ml.) were 8 x 10(6) in controls and 3 x 10(6) in shock wave, 3 x 10(7) in ultrasonic, 4 x 10(5) in electrohydraulic, 8 x 10(6) in pneumatic, 5 x 10(4) in holmium:YAG and 1 x 10(6) in pulsed dye laser lithotripsy cases (p <0.001). The rate of bacterial sterilization was 50% for holmium:YAG lithotripsy treated stones versus 0% for each of the other cohorts (p <0.01). CONCLUSIONS: P. mirabilis viability varies among lithotrites. The photothermal mechanism of holmium:YAG lithotripsy is antibacterial.

Holmium:YAG laser lithotripsy: A dominant photothermal ablative mechanism with chemical decomposition of urinary calculi.

BACKGROUND AND OBJECTIVE: Evidence is presented that the fragmentation process of long-pulse Holmium:YAG (Ho:YAG) lithotripsy is governed by photothermal decomposition of the calculi rather than photomechanical or photoacoustical mechanisms as is widely thought. The clinical Ho:YAG laser lithotriptor (2.12 microm, 250 micros) operates in the free-running mode, producing pulse durations much longer than the time required for a sound wave to propagate beyond the optical penetration depth of this wavelength in water. Hence, it is unlikely that shock waves are produced during bubble formation. In addition, the vapor bubble induced by this laser is not spherical. Thus the magnitude of the pressure wave produced at cavitation collapse does not contribute significantly to lithotripsy. STUDY DESIGN/MATERIALS AND METHODS: A fast-flash photography setup was used to capture the dynamics of urinary calculus fragmentation at various delay times following the onset of the Ho:YAG laser pulse. These images were concurrently correlated with pressure measurements obtained with a piezoelectric polyvinylidene-fluoride needle-hydrophone. Stone mass-loss measurements for ablation of urinary calculi (1) in air (dehydrated and hydrated) and in water, and (2) at pre-cooled and at room temperatures were compared. Chemical and composition analyses were performed on the ablation products of several types of Ho:YAG laser irradiated urinary calculi, including calcium oxalate monohydrate (COM), calcium hydrogen phosphate dihydrate (CHPD), magnesium ammonium phosphate hexahydrate (MAPH), cystine, and uric acid calculi. RESULTS: When the optical fiber was placed perpendicularly in contact with the surface of the target, fast-flash photography provided visual evidence that ablation occurred approximately 50 micros after the initiation of the Ho:YAG laser pulse (250-350 micros duration; 375-400 mJ per pulse), long before the collapse of the cavitation bubble. The measured peak acoustical pressure upon cavitation collapse was negligible (< 2 bars), indicating that photomechanical forces were not responsible for the observed fragmentation process. When the fiber was placed in parallel to the calculus surface, the pressure peaks occurring at the collapse of the cavitation were on the order of 20 bars, but no fragmentation occurred. Regardless of fiber orientation, no shock waves were recorded at the beginning of bubble formation. Ablation of COM calculi (a total of 150 J; 0.5 J per pulse at an 8-Hz repetition rate) revealed different Ho:YAG efficiencies for dehydrated calculus, hydrated calculus, and submerged calculus. COM and cystine calculi, pre-cooled at -80 degrees C and then placed in water, yielded lower mass-loss during ablation (20 J, 1.0 J per pulse) compared to the mass-loss of calculi at room temperature. Chemical analyses of the ablated calculi revealed products resulting from thermal decomposition. Calcium carbonate was found in samples composed of COM calculi; calcium pyrophosphate was found in CHPD samples; free sulfur and cysteine were discovered in samples composed of cystine samples; and cyanide was found in samples of uric acid calculi. CONCLUSION: These experimental results provide convincing evidence that long-pulse Ho:YAG laser lithotripsy causes chemical decomposition of urinary calculi as a consequence of a dominant photothermal mechanism.

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids.

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.

Holmium: YAG lithotripsy: photothermal mechanism.

OBJECTIVE: A series of experiments were conducted to test the hypothesis that the mechanism of holmium:YAG lithotripsy is photothermal. METHODS AND RESULTS: To show that holmium:YAG lithotripsy requires direct absorption of optical energy, stone loss was compared for 150 J Ho:YAG lithotripsy of calcium oxalate monohydrate (COM) stones for hydrated stones irradiated in water (17+/-3 mg) and hydrated stones irradiated in air (25+/-9 mg) v dehydrated stones irradiated in air (40+/-12 mg) (P < 0.001). To show that Ho:YAG lithotripsy occurs prior to vapor bubble collapse, the dynamics of lithotripsy in water and vapor bubble formation were documented with video flash photography. Holmium:YAG lithotripsy began at 60 microsec, prior to vapor bubble collapse. To show that Ho:YAG lithotripsy is fundamentally related to stone temperature, cystine, and COM mass loss was compared for stones initially at room temperature (approximately 23 degrees C) v frozen stones ablated within 2 minutes after removal from the freezer. Cystine and COM mass losses were greater for stones starting at room temperature than cold (P < or = 0.05). To show that Ho:YAG lithotripsy involves a thermochemical reaction, composition analysis was done before and after lithotripsy. Postlithotripsy, COM yielded calcium carbonate; cystine yielded cysteine and free sulfur; calcium hydrogen phosphate dihydrate yielded calcium pyrophosphate; magnesium ammonium phosphate yielded ammonium carbonate and magnesium carbonate; and uric acid yielded cyanide. To show that Ho:YAG lithotripsy does not create significant shockwaves, pressure transients were measured during lithotripsy using needle hydrophones. Peak pressures were <2 bars. CONCLUSION: The primary mechanism of Ho:YAG lithotripsy is photothermal. There are no significant photoacoustic effects.

Activated rate processes and a specific biochemical mechanism for explaining delayed laser induced thermal damage to the retina.

Laser induced thermal damage to the retina is investigated. The one step Arrhenius type thermal damage integral of Henriques is analyzed for its strengths and weaknesses. The zero-order activated rate process is shown to well represent the data for pulse durations greater than 10 µs. A zero-order biochemical damage mechanism involving free radical formation and thermal disruption of the melanosome's protein coat is proposed as the initial molecular process that leads to cellular damage which appears after a delay. Data are presented that show the photoactivation of melanin granule oxidative reactivity. This in vitro data is evidence for an important step in our hypothesized damage pathway. © 1999 Society of Photo-Optical Instrumentation Engineers.

Holmium:yttrium-aluminum-garnet lithotripsy efficiency varies with stone composition.

OBJECTIVES: To test the hypothesis that holmium:yttrium-aluminum-garnet (YAG) lithotripsy efficiency varies with stone composition. METHODS: Single pulses of holmium:YAG energy were delivered using 272-, 365-, 550-, and 940-microm optical fibers to human calculi composed of calcium oxalate monohydrate (COM), calcium hydrogen phosphate dihydrate (CHPD), cystine, magnesium ammonium phosphate hexohydrate (MAPH), and uric acid. Energy/pulse settings were 0.2, 0.5, 1.0, and 1.5 J. Stone crater width and depth were characterized with reflectance light microscopy. RESULTS: For similar energies overall MAPH yielded the deepest and widest craters. CHPD, cystine, and uric acid yielded craters of intermediate width and depth. COM yielded the smallest craters. Within any given composition, increased pulse energy yielded craters of increased width and depth. CONCLUSIONS: Holmium:YAG lithotripsy efficiency varies with stone composition. The rank order of crater size appears to correlate with thermal threshold for each composition. Increased holmium:YAG energy produces larger craters for all compositions.

Holmium:YAG lithotripsy: photothermal mechanism converts uric acid calculi to cyanide.

PURPOSE: Holmium:YAG lithotripsy fragments stones through a photothermal mechanism. Uric acid when heated is known to be converted into cyanide. We test the hypothesis that holmium: YAG lithotripsy of uric acid calculi produces cyanide. MATERIALS AND METHODS: Human calculi of known uric acid composition were irradiated with holmium:YAG energy in water. Stones received a total holmium:YAG energy of 0 (control), 0.1, 0.25, 0.5, 0.75, 1.0 or 1.25 kJ. The water in which lithotripsy was performed was analyzed for cyanide concentration. A graph was constructed to relate holmium:YAG energy to cyanide production. RESULTS: Holmium:YAG lithotripsy of uric acid calculi in vitro produced cyanide consistently. Cyanide production correlated with total holmium:YAG energy (p <0.001). CONCLUSIONS: Holmium:YAG lithotripsy of uric acid calculi risks production of cyanide. This study raises significant safety issues.

Holmium:YAG lithotripsy efficiency varies with energy density.

PURPOSE: We test the hypothesis that holmium:YAG lithotripsy efficiency varies with optical fiber size and energy settings (energy density). MATERIALS AND METHODS: The 272, 365, 550 and 940 microm. optical fibers delivered 1 kJ. total holmium:YAG energy to calcium oxalate monohydrate calculi at energy output/pulse of 0.2 to 1.5 J. Stone mass loss was measured for each fiber energy setting. Stone crater width was characterized for single energy pulses. Fiber energy outputs were compared before and after lithotripsy. RESULTS: Stone mass loss correlated inversely with optical fiber diameter (p <0.05). Stone loss correlated with energy/pulse for the 365, 550 and 940 microm. fibers (p <0.001). The 272 and 365 microm. fibers yielded equivalent stone loss at 0.2 and 0.5 J. per pulse. At energies of 1.0 J. per pulse or greater the 272 microm. optical fiber was prone to damage, and yielded reduced energy output and stone loss compared to the 365 microm. fiber (p <0.01). Stone crater width for single pulse energies correlated with energy settings for all fibers (p <0.001). CONCLUSIONS: Lithotripsy efficiency with the holmium:YAG laser depends on pulse energy output and diameter of the optical delivery fiber, implying that lithotripsy efficiency correlates with energy density. The 365 microm. fiber is indicated for most lithotripsy applications. The 272 microm. fiber is susceptible to damage and inefficient energy transmission at energies of 1.0 J. per pulse or greater. The 272 microm. fiber is indicated at energies of less than 1.0 J. per pulse for small caliber ureteroscopes or when maximal flexible ureteroscope deflection is required.

Holmium:YAG percutaneous nephrolithotomy: the laser incident angle matters.

PURPOSE: Laser physics dictate that maximal radiant exposure occurs when the laser strikes the target at a normal incidence. Since the renal collecting system often limits nephroscope movements during percutaneous nephrolithotomy, the laser-calculus incidence angle may vary. We have observed during holmium:YAG percutaneous nephrolithotomy that the side firing fiber more easily approaches a normal incidence compared to the end firing fiber. We test the hypothesis that holmium:YAG percutaneous nephrolithotomy is faster with a side firing fiber compared to an end firing fiber. MATERIALS AND METHODS: Consecutive holmium:YAG percutaneous nephrolithotomy cases were studied retrospectively. The calculus size and composition, surgical time, fragmentation speed (size/time) and stone-free rates were compared between 11 patients treated with end and 8 treated with side firing fibers. RESULTS: When the end and side firing groups were compared, calculus sizes (mean plus or minus standard deviation) were 22 +/- 13 versus 48 +/- 17 mm. (p = 0.004), calcium oxalate monohydrate incidence was 55 versus 75% (p = 0.3), surgical times were 168 +/- 87 versus 124 +/- 51 minutes, computed fragmentation speeds were 0.15 +/- 0.09 versus 0.43 +/- 0.15 mm. per minute (p = 0.0009) and stone-free rates were 73 versus 88% (p = 0.4), respectively. CONCLUSIONS: The side firing fiber is faster than the end firing fiber for holmium:YAG percutaneous nephrolithotomy. These results are consistent with principles of laser physics.

Holmium:YAG laser-induced damage to guidewires: experimental study.

The holmium:YAG laser fragments stones of all compositions effectively. However, damage to ureteral guidewires by the laser has been described, including in one of our own patients, in whom such damage resulted in morbidity. The purpose of this study was to characterize the interaction of Ho:YAG energy with guidewires in vitro. Seven ureteral guidewires were tested in a waterbath. The 365-microm Ho:YAG laser fiber was placed at defined distances (0, 1, 2, 4, and 5 mm) from the guidewire. All guidewires were tested at angles of 0 degrees, 45 degrees, and 70 degrees from normal incidence. The minimum energy setting that resulted in structural damage to the guidewires was detected by endoscopic video monitoring. All guidewires were susceptible to Ho:YAG laser damage at modest energy settings. The energy required to produce visual damage varied inversely with the square of the distance of the laser fiber from the guidewire. At a distance of 5 mm, none of the guidewires was damaged, even at energy settings of 2.8 J (the maximum output from the laser). The energy required to induce guidewire damage varied with the inverse of the cosine of the incident angle. The results demonstrate that no guidewire is immune from Ho:YAG laser damage when the fiber and guidewire are in contact. Caution must be exercised when operating the Ho:YAG laser near a guidewire, and guidewire integrity should be assured by the surgeon. Generally, the energy required to induce guidewire damage exceeded lithotripsy levels at distances >1 mm and with higher incident angles, implying a reasonable margin of safety during ureteroscopy. The pattern of energy thresholds required to induce damage with respect to distance and incident angle suggests that the mechanism of Ho:YAG lithotripsy is thermal.

Comparison of peroxidases in the retina, choroid and red blood cells.

Most of the peroxidase activity in the bovine retina is specific to glutathione (GSH) while the choroid contains both GSH peroxidases and ascorbate peroxidase. The GSH peroxidase was clearly separated from ascorbate peroxidase on a cation exchange column. The nonspecific peroxidase activity of hemoproteins accounts for the peroxidase activity detected by ascorbate oxidation. All choroidal hemoproteins contain subunits very similar to those of hemoglobin. The absence of ascorbate peroxidase in the retina indicates that the protective effect of ascorbate against photic injury is not due to its reaction with retinal peroxidase. Therefore removal of H2O2 by ascorbate peroxidase activity in the choroid could be a significant factor in studies where serum ascorbate concentrations are artificially raised far above the normal level concurrent to a very small rise in retinal ascorbate concentration.

Kinetic properties of light-dependent ascorbic acid oxidation by melanin.

The kinetic properties of ascorbic acid oxidation by light-activated melanin granules demonstrate the presence of a specific reactive site on the melanin granule saturable by ascorbic acid. Increased light intensity increased the Vmax and reduced the Km of this reaction, indicating increased affinity of the active site for ascorbic acid. The kinetics of this reaction are not markedly changed in a reduced-oxygen environment. Ascorbic acid oxidation is competitively inhibited by isoascorbic acid, an epimer of ascorbic acid, while other tested reducing agents are inactive. The Ki for isoascorbic acid is 1 mM, about the same as the Km of ascorbic acid.

Strategies for 3-D visualization of ocular structures.

The ability to visualize noninvasively the internal structure of the body has been widely utilized in clinical and research applications. Current tomographic structural imaging modalities, i.e., x-ray computed tomography (CT) and magnetic resonance imaging, produce image data which can be processed to reveal the three-dimensional surface of an internal object using surface rendering, or alternatively, the surface and internal details of the object using volume rendering. To take full advantage of these techniques, the acquisition and preparation of the tomographic data must be optimized for the imaging modality, for the target tissue, and for the specific visualization needs of the structure of interest. Strategies for optimizing each of these parameters are presented using, as an example, an x-ray CT study of a bony orbital fracture.

Sodium-dependent ascorbic and dehydroascorbic acid uptake by SV-40-transformed retinal pigment epithelial cells.

The present data confirmed previous studies with other cell types that ascorbic acid and dehydroascorbic acid are transported through different transporters into SV-40-transformed retinal pigment epithelial cells. These experiments were performed on cells grown on 96-well culture plates. Ascorbic acid was taken up into the cell by a high-affinity transporter with Km = 0.041 mmol/l and a low Vmax of 2.74 pmol/min/well. Dehydroascorbic acid was taken up by a low-affinity transporter with Km = 5.67 mmol/l; however, the Vmax was 325.5 pmol/min/well. The uptake of both ascorbic acid and dehydroascorbic acid was dependent on the sodium concentration. The uptake of ascorbic acid does not involve oxidation-reaction steps because the uptake of [14C]-ascorbate was unaffected by the presence of an excess amount of unlabelled dehydroascorbic acid.

Oxidation of ascorbic acid as an indicator of photooxidative stress in the eye.

When whole retinal pigmented epithelium (RPE) cells isolated from bovine eyes are incubated with 14C-labeled ascorbic acid and exposed to a visible laser, the ascorbic acid is oxidized to dehydro-L-ascorbic acid (DHA). The amount of ascorbic acid which is oxidized is proportional to the radiant exposure of the sample (i.e. the total amount of radiation per unit area delivered over the exposure time). Blue light is more effective than red light in driving the reaction. The amount of label appearing in the DHA fraction is increased if unlabeled DHA is present in the reaction mixture, indicating that some redox cycling of ascorbate is occurring in the RPE cells. The ascorbic acid oxidizing activity does not depend on intact cells, is not inactivated by heating the cells to 80 degrees C, and appears to reside mainly in the subcellular fraction which contains melanin pigment granules. The ascorbic acid oxidation may be caused by free radicals formed when melanin is illuminated with light. This reaction appears to be a useful method for quantifying the production of free radicals during photooxidative stress.