Improved conjunctival microcirculation in diabetic retinopathy patients with MTHFR polymorphisms after Ocufolin™ Administration

Abstract

Purpose: To investigate conjunctival microvascular responses in patients with mild diabetic retinopathy (MDR) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms (D + PM) after administration of Ocufolin™, a medical food containing 900 μg l-methylfolate (levomefolate calcium or [6S]-5-methyltetrahydrofolic acid, calcium salt), methylcobalamin, and other ingredients.

Methods: Eight D + PM patients received Ocufolin™ for six months (6 M). Bulbar conjunctival microvasculature and microcirculation metrics, including vessel diameter (D), axial blood flow velocity (Va), cross-sectional blood flow velocity (Vs), flow rate (Q), and vessel density (VD, Dbox), were measured at baseline, 4 M, and 6 M.

Results: The mean age was 54 ± 7 years. No significant demographic differences were found. Conjunctival microcirculation, measured as Va, Vs, and Q was significantly increased at 4 M and 6 M, compared to baseline. Va was 0.44 ± 0.10 mm/s, 0.58 ± 0.13 mm/s, 0.59 ± 0.13 mm/s in baseline, 4 M, and 6 M, respectively (P < 0.01). Similarly, Vs was 0.31 ± 0.07 mm/s, 0.40 ± 0.09 mm/s, 0.41 ± 0.09 mm/s in baseline, 4 M, and 6 M, respectively (P < 0.05). Q was 107.8 ± 49.4 pl/s, 178.0 ± 125.8 pl/s, 163.3 ± 85.8 mm/s in baseline, 4 M, and 6 M, respectively (P < 0.05). The VD at 6 M was significantly higher than that at baseline (P = 0.017). Changes of D were positively correlated with changes of Va, Q, and VD. Effects of MTHFR and haptoglobin polymorphisms on the improvements of conjunctival microcirculation and microvasculature were found.

Conclusions: Ocufolin™ supplementation improves conjunctival microcirculation in patients with diabetic retinopathy and common folate polymorphisms.

Keywords: Conjunctival microcirculation; Conjunctival microvasculature; Diabetes mellitus; Diabetic retinopathy; Functional slit-lamp biomicroscopy; Haptoglobin; MTHFR; Medical food; l-methylfolate.

Fig. 1.. Calculation of the bulbar conjunctival vascular diameter and blood flow velocity.

Video clips of the bulbar conjunctiva blood flow were captured using FSLB and processed to measure blood flow velocity. (A) The first frame of the video clip was utilized for registering all frames to compensate for the eye motion. (B) Using custom software, the vascular walls were outlined and marked in green and yellow lines for measuring the vessel diameter. (C) By measuring the image intensity within the locations defined by the vascular walls, an intensity profile along the centerline (red line in image B) between these walls was generated for each frame in the video clip. Using all intensity profiles (Y-axis) of all frames over time (X-axis) in the video clips, a space-time image was obtained and used to calculate the motion of the cluster of erythrocytes over time (i.e., blood flow velocity). The slopes of the bands (i.e., moving distance over time) were manually outlined (marked in red lines in Image C) and calculated as axial blood flow velocity.

Fig 2.. Image processing to extract the microvascular network.

(A) The raw image with an image size of 5,184 × 3,456 pixels was resized to 1,024 × 683 pixels. (B) Microvascular map after a series of morphological opening operations. (C) Segmented vessels using a binary process. (D) Skeletonized image acquired from the cropped images 512 × 512 pixels with a field of view 7.87 ×7.87 mm, which was for fractal analysis to obtain vessel density (VD) using box-counting.

Fig 3.. The changes of conjunctival microvasculature and microcirculation after the intake of the Ocufolin™ in D+PM patients.

The measurements were taken at baseline, 4M, and 6M after intake of the medical food. Conjunctival microcirculation measured as Va (A), Vs (B), and Q (C) in patients with D+PM were significantly increased at 4M and 6M, compared to baseline (P < 0.05). There were no significant differences in the D (D) in the D+PM group among visits (P > 0.05). The VD (E) at 6M in the D+PM group was significantly higher than that at baseline (P = 0.017). D+PM, mild diabetic retinopathy patients with methylenetetrahydrofolate reductase polymorphisms; Va, axial velocity; Vs, cross-sectional velocity; Q, flow rate; VD, vessel density.

Fig. 4.. The effect of MTHFR polymorphisms and HP genotypes on the improvements of conjunctival microcirculation and microvasculature in patients with D+PM during the Ocufolin™ administration.

Va and Vs at 6M were significantly increased over time at all genotypes subgroups (all P < 0.05). D and Q at 6M in patients with HP2-2 genotype were decreased when compared to baseline. D, Q, and VD at 6M in patients with A1298C, C677T, and A1298C, and HP1-1/1-2 were increased significantly over time (all P < 0.05). MTHFR, methylenetetrahydrofolate reductase; HP, haptoglobin; D+PM, mild diabetic retinopathy patients with methylenetetrahydrofolate reductase polymorphisms; Va, axial blood flow velocity; Vs, cross-sectional blood flow velocity; D, vessel diameter; Q, flow rate, VD, vessel density.

Fig.5.. Correlations among changes at 6M after the intake of Ocufolin™ medical food.

In patients with D+PM, the changes of Va and Q at 6M were positively related to the change of D (P < 0.05). However, the changes of Vs and VD at 6M were not related to the changes in D (P > 0.05). D+PM, mild diabetic retinopathy patients with methylenetetrahydrofolate reductase polymorphisms; D, vessel diameter; Va, axial blood flow velocity; Vs, cross-sectional blood flow velocity; Q, flow rate; VD, vessel density.