Effect of vitamin B12 supplementation on retinal lesions in diabetic rats.

Purpose: Diabetic retinopathy (DR) is the most common complication of diabetes involving microvasculature and neuronal alterations in the retina. Previously, we reported that vitamin B12 deficiency could be an independent risk factor for DR in humans. However, the effect of vitamin B12 supplementation in experimental DR is unknown. Thus, in this study, we investigated the impact of dietary supplementation of vitamin B12 on retinal changes in diabetic rats. Methods: Diabetes was induced in 2-month-old Sprague-Dawley rats and maintained for 4 months. One group of diabetic rats were fed normal levels of vitamin B12, and one group double the quantity of vitamin B12 (50 µg/kg diet). Vitamin B12 and homocysteine levels in the plasma were analyzed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC), respectively. At the end of 4 months of experimentation, the eyeballs were collected. Retinal changes were analyzed with hematoxylin and eosin (H&E) staining, immunoblotting, and immunofluorescence methods. Results: Dietary supplementation of vitamin B12 had no effect on food intake, bodyweight, fasting blood glucose, and plasma homocysteine levels in the diabetic rats. However, vitamin B12 supplementation prevented loss of rhodopsin, and overexpression of VEGF, and completely prevented overexpression of HIF1α, GFAP, and endoplasmic reticulum (ER) stress markers (GRP78, ATF6α, XBP1, CHOP, and caspase 12) in the diabetic rat retina. Further, vitamin B12 ameliorated apoptosis in the retina as shown with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and prevented retinal thinning. Conclusions: Vitamin B12 supplementation of diabetic rats appeared to be beneficial by circumventing retinal hypoxia, VEGF overexpression, and ER stress-mediated cell death in the retina. The present study adds another potential therapeutic strategy of vitamin B12 in diabetes.

A two-step method for identifying photopigment opsin and rhodopsin gene sequences underlying human color vision phenotypes.

Purpose: To present a detailed, reliable long range-PCR and sequencing (LR-PCR-Seq) procedure to identify human opsin gene sequences for variations in the long wavelength-sensitive (OPN1LW), medium wavelength-sensitive (OPN1MW), short wavelength-sensitive (OPN1SW), and rhodopsin (RHO) genes. Methods: Color vision was assessed for nine subjects using the Farnsworth-Munsell 100 hue test, Ishihara pseudoisochromatic plates, and the Rabin cone-contrast threshold procedure (ColorDX, Konan Medical). The color vision phenotypes were normal trichromacy (n = 3), potential tetrachromacy (n = 3), dichromacy (n = 2), and unexplained low color vision (n = 1). DNA was isolated from blood or saliva and LR-PCR amplified into individual products: OPN1LW (4,045 bp), OPN1MW (4,045 bp), OPN1SW (3,326 bp), and RHO (6,715 bp). Each product was sequenced using specific internal primer sets. Analysis was performed with Mutation Surveyor software. Results: The LR-PCR-Seq technique identified known single nucleotide polymorphisms (SNPs) in OPN1LW and OPN1MW gene codons (180, 230, 233, 277, and 285), as well as those for lesser studied codons (174, 178, 236, 274, 279, 298 and 309) in the OPN1LW and OPN1MW genes. Additionally, six SNP variants in the OPN1MW and OPN1LW genes not previously reported in the NCBI dbSNP database were identified. An unreported poly-T region within intron 5(c.36+126) of the rhodopsin gene was also found, and analysis showed it to be highly conserved in mammalian species. Conclusions: This LR-PCR-Seq procedure (single PCR reaction per gene followed by sequencing) can identify exonic and intronic SNP variants in OPN1LW, OPN1MW, OPN1SW, and rhodopsin genes. There is no need for restriction enzyme digestion or multiple PCR steps that can introduce errors. Future studies will combine the LR-PCR-Seq with perceptual behavior measures, allowing for accurate correlations between opsin genotypes, retinal photopigment phenotypes, and color perception behaviors.

Rhodopsin in plasma from patients with diabetic retinopathy - development and validation of digital ELISA by Single Molecule Array (Simoa) technology.

BACKGROUND: Diabetic retinopathy (DR) is the most frequent cause of blindness among younger adults in the western world. No blood biomarkers exist to detect DR. Hypothetically, Rhodopsin concentrations in blood has been suggested as an early marker for retinal damage. The aim of this study was therefore to develop and validate a Rhodopsin assay by employing digital ELISA technology, and to investigate whether Rhodopsin concentrations in diabetes patients with DR are elevated compared with diabetes patients without DR. METHODS: A digital ELISA assay using a Simoa HD-1 Analyzer (Quanterix©, Lexington, MA 02421, USA) was developed and validated and applied on a cohort of diabetes patients characterised with (n=466) and without (n=144) DR. RESULTS: The Rhodopsin assay demonstrated a LOD of 0.26ng/l, a LLOQ of 3ng/l and a linear measuring range from 3 to 2500ng/l. Total CV% was 32%, 23%, 19% and 17% respectively at the following Rhodopsin concentrations: 1, 3, 5 and 13ng/l. Recovery was 17%, 34%, 51% and 55% respectively at Rhodopsin concentrations of 2, 10, 50 and 250ng/l. There was no statistically significant difference in the plasma concentration of Rhodopsin between the diabetes patients with or without DR, but significantly increased number of DR patients having concentrations above the LOD. CONCLUSION: We developed and validated a digital ELISA method for quantification of Rhodopsin in plasma but found no statistically significant difference in the plasma concentration of Rhodopsin between diabetes patients with DR compared to diabetes patients without DR, though significantly more DR patients had values above the LOD.

Real-time quantitative PCR measurement of circulatory rhodopsin mRNA in healthy subjects and patients with diabetic retinopathy.

Diabetic retinopathy is the commonest complication of diabetes and is the biggest single cause of registered blindness in the UK. No biochemical tests exist to determine the precise state and rate of change of the eyes in the diabetic patient. In the present study, using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we measured mRNA encoding the retina-specific pigment protein rhodopsin (RHO) in the peripheral blood of healthy individuals (n = 20) and diabetic patients (n = 46) with and without retinopathy. Beta-actin mRNA was also assayed and results are expressed as a ratio of RHO to beta-actin mRNA. Peripheral blood was taken by venipucture directly into PAXgene Blood RNA collection tubes and RNA extracted by use of the PAXgene Blood RNA extraction kit, as per the manufacturer's (Qiagen) instructions. Diabetic patients were divided into three groups defined by the severity of retinopathy as assessed by fundoscopy: A, diabetic without retinopathy; B, background retinopathy; and C, preproliferative retinopathy. Medians of the ratios between groups were compared. RHO mRNA was successfully detected and quantified in peripheral blood in all healthy and diabetic groups, with levels shown to be significantly higher in diabetic patients than in healthy controls (2.54 x 10(-5) vs. 1.29 x 10(-5); P = 0.002). Significant differences in RHO mRNA levels were also seen between healthy control subjects and diabetic groups A (2.52 x 10(-5); P = 0.022), B (1.98 x 10(-5); P = 0.028), and C (5.08 x 10(-5); P = 0.002). The results suggest that there is an increase in circulatory RHO mRNA with the severity of diabetic retinopathy.