Kiyomi Arai

Effects of astaxanthin on antioxidation in human aqueous humor

We evaluated the antioxidative effects of astaxanthin through the changes in superoxide scavenging activity, levels of hydrogen peroxide and total hydroperoxides in human aqueous humor. The study subjects were 35 patients who underwent bilateral cataract surgery on one side before and the other side after intake of astaxanthin (6 mg/day for 2 weeks). Their aqueous humor was taken during the surgery and subjected to measurements of the three parameters. After astaxanthin intake, the superoxide scavenging activity was significantly (p<0.05) elevated, while the level of total hydroperoxides was significantly (p<0.05) lowered. There was a significant negative correlation between the superoxide scavenging activity and the level of total hydroperoxides (r = −0.485, p<0.01), but no correlations between the hydrogen peroxide level and the other two parameters. Astaxanthin intake clearly enhanced the superoxide scavenging activity and suppressed the total hydroperoxides production in human aqueous humor, indicating the possibility that astaxanthin has suppressive effects on various oxidative stress-related diseases.

Levels of mature cross-links and advanced glycation end product cross-links in human vitreous.

PURPOSE To determine the levels of pyridinoline and deoxypyridinoline, two mature enzymatic cross-links, and pentosidine, an advanced glycation end product (AGE) cross-link, in human vitreous and to investigate the correlations among the cross-links and the effects of aging and diabetes mellitus (DM) on the levels of the cross-links. METHODS Forty-five vitreous samples were collected from 32 eyes undergoing vitrectomy for diabetic retinopathy (DM group) and 13 eyes (control group) from age- and sex-matched patients with idiopathic macular hole or epiretinal membrane with no systemic conditions. The levels of the cross-links were determined using high-performance liquid chromatography after acid hydrolysis and pre-treatment with SP-Sephadex. RESULTS The levels of pentosidine, pyridinoline, and deoxypyridinoline were 27.3 +/- 23.1(mean +/- standrard deviation) pmol/ml (n = 45), 79.0 +/- 40.2 (mean +/- standrard deviation) ng/ml (n = 43), and 54.0 +/- 9.5 ng/ml (n = 32), respectively. When the vitreous samples from DM and the controls were compared, a significant difference (p < 0.05) was found in the level of pentosidine but not in the levels of pyridinoline or deoxypyridinoline. No significant correlations were found between age and the cross-links. Significant correlations (p < 0.01) were found among the cross-links. CONCLUSIONS The results indicate that mature cross-link substances exist in human vitreous. The results also suggest that glycation may occur in the vitreous after mature cross-links form and result in the formation of AGE cross-links. In human vitreous from patients with DM, increased levels of AGE cross-links may stabilize the formation of mature cross-links but did not increase mature cross-links.

Pentosidine and autofluorescence in lenses of diabetic patients

Pentosidine, an advanced glycation endproduct, may cause the increased fluorescence found in the lenses of diabetic patients. We measured the autofluorescence in human lenses, in vitro, and quantified the pentosidine to examine its relationship to the autofluorescence. Lens autofluorescence was higher in diabetic than non-diabetic subjects; pentosidine quantities were significantly higher in the diabetic than the non-diabetic group indicating that pentosidine was involved in the greater intensity of autofluorescence in the lenses of patients with diabetes mellitus.

Free radicals in the aqueous humor of patients with glaucoma

Purpose: To clarify the presence of oxidative stress in glaucoma and discuss whether free radicals contribute to neovascular glaucoma (NVG) and non-NVG. Methods: Two patient groups were formed: the NVG group (n = 10) and the non-NVG group (n = 17). Free radicals in aqueous humor were detected by measuring the electron spin resonance (ESR). To identify free radical species, either superoxide dismutase (SOD) or catalase was added to the aqueous humor and compared with aqueous humor in which SOD or catalase was not added. Results: In the NVG group, free radical waveforms were detected that differed from ascorbate-free radical (AFR) in all cases. Under SOD was added, the characteristic waveforms disappeared and the AFR of a specific waveform appeared. After catalase was added, the waveforms were unchanged. In the non-NVG group, the AFR of specific waveforms were detected in all cases. In 4 cases, the waveforms detected the presence of a trace of superoxide. Conclusions: In the NVG group, superoxides were detected, suggesting that superoxide scavenging activity was decreased markedly. L-ascorbic acid likely has an antioxidative function in the non-NVG group, suggesting that the aqueous humor in the NVG group was under higher oxidative stress compared with the non-NVG group.

Correlation between the matrix metalloproteinase-9 activity and chondroitin sulfate concentrations in tear fluid after laser in situ keratomileusis.

Aims: The aim of the present study was to evaluate the correlation between matrix metalloproteinase-9 (MMP-9) and chondroitin sulfate (CS) concentrations in human tear fluid following laser in situ keratomileusis (LASIK). Methods: Twelve eyes from six patients who had no ocular complaints except for refractive errors, and who had undergone LASIK, were enrolled in this study. We measured the concentrations of chondroitin 4 sulfate (C4S), chondroitin 6 sulfate (C6S), and MMP-9 activity with an enzyme-linked immunosorbent assay and an enzyme immunocapture activity assay preoperatively and postoperatively on days 1 and 4, week 1, and at 1 and 3 months. Results: Although the preoperative MMP-9 activity and the C4S concentration were highly correlated (r = 0.900; P < 0.001), they were not postoperatively correlated at month 1. Although the preoperative MMP-9 activity and the C6S concentration were highly correlated (r = 0.885; P < 0.001), they were not postoperatively correlated at either week 1 or months 1 and 3. Conclusions: The correlation appeared to be collapsed by LASIK, and it did not recover to the preoperative score at 3 months post-surgery. Our study indicates that the corneal wound healing was not terminated at 3 months following LASIK.

Relationship between Pentosidine and Pyridinoline Levels in Human Diabetic Cataract Lenses

The relationship between the levels of two different crosslink compounds, pentosidine and pyridinoline, in human diabetic cataract lenses was investigated to elucidate the pathogenic mechanism of diabetic cataract. Subjects were classified into diabetes mellitus (DM) group and non-DM group according to the presence or absence of DM. The levels of the crosslink compounds were determined using high-performance liquid chromatography and spectrofluorometry after acid hydrolysis. In the non-DM group the pentosidine level was significantly and positively correlated with the pyridinoline level and age. In the DM group the pentosidine level was not significantly correlated with either pyridinoline level or age. Pyridinoline levels and age were not significantly correlated in either group. The increase in crosslink compounds due to glycation and the relationship between the compounds are changed in DM lenses.

Photorefractive keratectomy: measuring the matrix metalloproteinase activity and chondroitin sulfate concentration in tear fluid

We herein report the case of a 20-year-old man who underwent a photorefractive keratectomy (PRK). We measured matrix metalloproteinase-9 (MMP-9) activity and chondroitin 4 sulfate and chondroitin 6 sulfate concentrations in tear fluid. Tear fluid was collected pre-operatively via microcapillary tube, and was collected postoperatively on the first and fourth days, and after one week, one month, three months, and six months. Samples were formulated by dilution with 200 μL of saline. MMP-9 activity was analyzed by an enzyme immunocapture activity assay, and the concentrations of chondroitin sulfate were analyzed by enzyme-linked immunosorbent assay. No complications were observed after surgery, except for a minimal subepithelial haze. Although MMP-9 activity changed on the fourth postoperative day, the activity changed only minimally at this time. Chondroitin 4 sulfate concentrations in tear fluid increased dramatically from one week to one month, decreased transiently at three months, and increased by six months. The chondroitin 6 sulfate concentration did not normalize within one week, and decreased from one week to three months compared with the preoperative score, and was close to the preoperative score at six months. We conclude that corneal wound healing was still incomplete six months after PRK, and chondroitin 4 sulfate appears to be critical in this process.

The effect of astaxanthin on vascular endothelial growth factor (VEGF) levels and peroxidation reactions in the aqueous humor

We explored the effect of astaxanthin on vascular endothelial growth factor in the aqueous humor, by measuring vascular endothelial growth factor levels and oxidation-related parameters, including O2•− scavenging activity, H2O2 level, and total hydroperoxide level in the aqueous humor, obtained from 35 patients before and after astaxanthin administration. We evaluated the relationship between vascular endothelial growth factor and the oxidation-related parameters as well as the patient’s diabetic status, age, and sex. Vascular endothelial growth factor levels did not change significantly but O2•− scavenging activity and total hydroperoxide level significantly (p<0.05) increased and decreased, respectively. Both pre- and post- astaxanthin intake, vascular endothelial growth factor and total hydroperoxide levels were positively correlated (Pearson: r = 0.42, p<0.05; r = 0.55, p<0.01, respectively). Analysis of vascular endothelial growth factor levels and O2•− scavenging activities gave a negative correlation but only pre-astaxanthin intake (r = −0.37, p<0.05). Differences in levels pre- and post-astaxanthin only showed association between vascular endothelial growth factor and total hydroperoxide (r = 0.49, p<0.01) analyzed by multiple linear regression. Using multivariate analysis, pre-astaxanthin vascular endothelial growth factor level was associated with two factors of total hydroperoxide and O2•− scavenging activity (r = 0.49, p<0.05), and post-astaxanthin vascular endothelial growth factor level with two factors of total hydroperoxide and sex (r = 0.60, p<0.01). Astaxanthin intake may have affected vascular endothelial growth factor level through its antioxidant effects by increasing O2•− scavenging activity and suppressing peroxide production.