Improving diabetic and hypertensive retinopathy with a medical food containing L-methylfolate: a preliminary report

Background Homocysteine and vitamin D may play a role in the development of diabetic and hypertensive retinopathy in patients with diabetes mellitus (DM) and hypertension. Supplementing food with L-methylfolate and vitamin D theoretically may improve diabetic and hypertensive retinopathy, however, the outcome of these nutritional approaches has not been fully examined. A retrospective case review was done of cases of retinopathy reversal in patients on Ocufolin™ and a similar nonprescription multivitamin, Eyefolate™. In this study, they were administered L-methylfolate (2.7 mg and 3.0 mg, respectively) and vitamin D3 (4500 IU each). These dosages are significantly above the RDA but well below levels associated with toxicity. Case presentation Seven patients had nonproliferative diabetic retinopathy (NPDR) and some of them had hypertension. One patient had only hypertensive retinopathy. All patients were instructed to take Ocufolin™ medical food as a food supplement. Baseline genetic testing for MTHFR polymorphisms was conducted. Fundus photography was used to document the fundus condition of the enrolled eyes in 8 NPDR patients at the initial and follow-up visits. Microaneurysms (MA) and exudates were observed to be improved in some trial patients. All subjects had one or more MTHFR polymorphisms. All had diabetic retinopathy, hypertensive retinopathy, or both. MAs were resolved, and exudates were decreased in 8/8 cases after taking the medical food. Retinal edema was found in 2/8 cases and improved or resolved in both cases after taking the medical food or the supplement. The best corrected visual activity was stable or improved in 8/8 cases. Conclusion We report a series of diabetic and hypertensive retinopathy cases with MTHFR polymorphisms and the improvement of retinal microvasculature (mainly MAs) in serial fundus photography after taking a medical food or supplement containing L-methylfolate and vitamin D. It appears that the use of nutritional supplements and medical foods containing L-methylfolate and vitamin D may be effective in facilitating the improvement of diabetic and hypertensive retinopathy.

response, deregulation of growth factors, and increased vascular permeability [6]. The current standard of care treatments is the injection of anti-VEGF drugs and the use of laser photocoagulation to slow vision loss and occasionally improve vision. However, these treatments are costly, invasive, stressful, and time-consuming for both doctors and patients. Both are often necessary for more advanced DR, which already pose a threat to vision but are generally avoided in the earlier stages of the disease. Hence, beyond good control of blood sugar and blood pressure, less invasive complementary treatments are needed to slow the progression of early DR. Ideally, such complementary treatments would not interfere with the current standard of care treatments. Previous studies found increased homocysteine associated with diabetic retinopathy [7,8]. The homocysteine affects vascular endothelial growth factor in the retina [9,10], which is also associated with the regulation of angiogenesis [11]. Reducing homocysteine is one such approach that addresses another driver of DR, without impairing anti-VEGF and laser therapies.
Vitamin D is a key regulator of several important metabolic functions [21,22]. Vitamin D insufficiency is associated with increased CRP, arterial stiffness, and endothelial dysfunction [22]. Vitamin D deficiency increases levels of parathyroid hormone (PTH), which increases insulin resistance and is associated with an increased incidence of diabetes [23]. Finally, vitamin D deficiency in type 2 diabetes patients increases the risk of DR [24]. The deficiency of vitamin D may play an important role in the progression and severity of DR [25][26][27].
Treating diabetic and hypertensive retinopathy with vitamin D and L-methylfolate may be helpful; however, the efficacy of these treatments has not been fully studied. Food supplementation is safe, simple, and inexpensive. A previous study reported that folic acid therapy reduces the risk of stroke [28]. Folic acid supplementation was also found to be associated with reduced risk of retinal microangiopathy in adults with hypertension complicated with DM [17]. Another study reported that the medical food Ocufolin™ may have a marked improvement in retinal artery occlusion outcome as late as 24 h post occlusion [29]. L-methylfolate (5-MTHF) more readily crosses the blood brain barrier and blood retinal barrier than folic acid and has less toxicity than folic acid, thus making it a more potent choice for retinal therapy than folic acid [30][31][32]. Here, we present the findings of 8 patients with diabetic and/or hypertensive retinopathy who took food supplements with supratherapeutic levels of L-methylfolate and vitamin D.

Methods
During a prospective study of the effects of medical food, Ocufolin™ on retinal blood flow in patients with diabetic retinopathy, visible improvements of baseline retinopathy were noted. This prompted a retrospective review of other patients treated with Ocufolin™ and a similar product, Eyefolate™ for visible improvements of retinopathy.
We reviewed 7 retrospectively identified cases from the Eye Center, Fayetteville, AR and 1 case from Bascom Palmer Eye Institute, University of Miami, Miami, FL. This study was performed from Oct 2013 to Dec 2018. The Washington Regional Institutional Review Board waived review of this retrospective case study of anonymized case data from the Fayetteville site, and the University of Miami Institutional Review Board approved the prospective study conducted at the University of Miami. Written informed consent to be included in this study was obtained from each study subject.
All subjects had been examined by experienced ophthalmologists. Every patient received a full ophthalmic examination including slit-lamp biomicroscopy, intraocular pressure (IOP) measurement, refraction, best corrected visual acuity (BCVA) and fundus indirect ophthalmoscopic examination, and fundus photography. Baseline diabetic retinopathy severity scores were determined by the retinal specialist, according to the International Clinical Diabetic Retinopathy Disease Severity Scale. Genetic testing was performed by MyGenetx Laboratory, LLC (Franklin, TN) or Quest Diagnostics. In addition, other blood tests were performed by Quest Diagnostics, including homocysteine, vitamin D, vitamin B12 and HbA1c. Standard fundus photos were taken using a Zeiss Visucam NM/FA at the Fayetteville Eye Center. A Topcon (50DX, Topcon Medical Systems, Inc., Oakland, NJ) fundus camera and a Zeiss Clarus fundus camera (Model 500, Carl Zeiss Meditec, Inc., Dublin, CA) were used at the Bascom Palmer Eye Institute.
Case 8 was one of the patients recruited in a prospective clinical study using Ocufolin™ medical food supplementation at the Bascom Palmer Eye Institute. This patient was imaged using the retinal function imager (RFI), an advanced ophthalmic imaging modality based on a fundus camera. The retinal blood flow velocity was measured. The detailed information and applications of RFI were reviewed previously [33]. Before the study, the pupil of this patient was dilated with 1% tropicamide. A field of view of 4.3 × 4.3 mm 2 (20 degrees) was used. The retinal blood flow velocity was measured in the arterioles and venules.

Results
Basic information from the 8 cases is presented in Table 1. All subjects were Caucasians and diagnosed with nonproliferative diabetic retinopathy (NPDR) and/ or hypertensive retinopathy (HR). Three were females and five were males.
Among them, 7 subjects took Eyefolate™ (3 subjects switched to Ocufolin™) and one subject took only Ocufo-lin™. Detailed information of these formulations is listed in Table 2. During the food supplement treatment period, 2 subjects had BCVA improvement and 3 subjects had mild BCVA decline ( Table 3). The remaining 3 subjects remained at the same BCVA. All subjects showed resolution of intra-retinal hemorrhages, microaneurysms (MA), and or the reduction of exudates on the fundus photograph. Detailed information of each case is presented below.

Case 1
A 67-year-old woman presented with bilateral NPDR and treated hypertension. BCVA was 20/40-2 OU at the first visit in April 2014. Fundus photos showed multiple MAs, mild dot and blot retinal hemorrhages with some exudates in the right eye (OD) (Fig. 1a). She was instructed to take 3 capsules of Eyefolate™ daily with food. In October 2015, the BCVA was 20/50 in OD, and 20/30 in the left eye (OS). Reduced MAs and retinal hemorrhages were observed ( Fig. 1b). At that visit, mild diabetic cystoid macular edema (CME) was found in the OCT image (Fig. 1d). In September 2018, the subject received bilateral intraocular lens (IOL) implantation surgery after which the BCVA improved to 20/30 in OD and 20/25 in OS. The right eye showed fewer  hemorrhages and MA (Fig. 1c) and less CME on OCT examination (Fig. 1e).

Case 2
An 82-year-old woman presented with diet-controlled type 2 diabetes and medication controlled hypertension. In June 2014, the BCVA in OD was hand movement (HM) due to an old macular scar and the BCVA in OS was 20/20. Intra-retinal hemorrhages and 2 small MAs were found in the fundus photo OS (Fig. 2a). She began 3 capsules of Eyefolate™ daily with breakfast. In October 2014, her BCVA remained HM in OD and 20/25 OS, but the hemorrhages resolved and only 1 faint MA was seen in the fundus photography in OS (Fig. 2b).  The MA was resolved in the OS fundus photo (Fig. 3c) in 2016.

Case 4
The fourth case was a 59-year-old man with an 11-year history of type 2 diabetes who had required insulin for the past 8 years. On his first visit in October 2014, the BCVA was 20/20 OU. Multiple small intra-retinal hemorrhages and MAs with exudates superotemporally were found OS as seen in the fundus image (Fig. 4a). He began 3 capsules of Eyefolate™ daily. In October 2015, the number of retinal hemorrhages and MAs declined OS (Fig. 4b), then he switched from Eyefolate™ to Ocufolin™. In April 2016, the number of hemorrhages and MAs continued to decline, and the exudates resolved OS (Fig. 4c).

Case 5
A 76-year-old nondiabetic man was examined with a 10 year history of medication controlled hypertension and dry age-related macular degeneration (AMD) (drusen) OU. In June 2014, multiple small MAs with exudates and one cotton wool spot was found OD as seen in the fundus photo (Fig. 5a) inferotemporal to small drusen. The retinopathy was likely initiated by a small BRVO, with hypertension as a continuing co-factor, the BCVA was 20/20 OU. He began to take 3 capsules of Eyefolate™ daily. In December 2014, resolution of the cotton wool spot, exudates, and MAs was found OD (Fig. 5b). In May 2018, smaller MAs and exudates reappeared in the original locations after he had self-discontinued the Eyefolate™ (Fig. 5c).

Case 6
The sixth case was a 56-

Case 7
The seventh case was a 64-year-old man with a 10 year history of medication controlled hypertension and newly diagnosed type 2 diabetes. In August 2018, a diabetic intra-retinal hemorrhage was found OS (Fig. 7a) and he then began to take Eyefolate™. By December 2018, the intra-retinal hemorrhage was resolved (Fig. 7b).

Case 8
The eighth case was a 68-year-old man with NPDR OS. Initially, he was diagnosed with mild NPDR OS (October 2017). The BCVA was 20/20 OU. A MA was apparent in the superonasal quadrant OS in 2017 (Fig. 8). He participated in a clinical trial of Ocufolin™ at the Bascom Palmer Eye Institute, University of Miami and was  11, 2018, Fig. 9).

Discussion and conclusions
DM and hypertension cause microvascular abnormalities, including DR and hypertensive retinopathy (HR) [14,[34][35][36][37]. Microvascular abnormalities, especially retinal arterial narrowing and retinal hemorrhages, have been used to monitor the impact on the microvasculature in the body as a whole, in the central nervous system, and small vessel white matter disease [38,39]. In addition, retinopathy poses a threat to vision and may cause blindness. Small vessel abnormalities and formation and turnover rates of MAs in the retina have been regarded as reliable predictors of the onset and progression of DR [28]. The newer MAs in successive fundus Our study presents a case series of patients with NPDR and HR and their findings after taking a medical food containing L-methylfolate and vitamin D. The primary findings were the resolution of retinal hemorrhages and MAs, as well as the reduction of exudates and macular edema. This suggests that a supplement or medical food containing therapeutic doses of L-methylfolate and vitamin D may be a safe and noninvasive addition to our treatments of NPDR and HR.
Both Eyefolate™ and Ocufolin™ contain L-methylfolate, which targets the ischemic consequences of reduced function polymorphisms of the MTHFR gene in patients with DM. Wicken et al. reported that the C667T was present in 40% of the Caucasian American population, 50% of Europeans and a lower percentage in African-Americans and Asian-Americans [41]. MTHFR C677T and A1298C polymorphisms lead to a reduction in the methylation of tetrahydrofolate, which results in decreased available L-methylfolate, methylcobalamin (methyl B12), and the elevation of homocysteine. These further lead to elevated blood pressure damaging the small vessels [42].
In a recent study of 24 patients with DM, plasma homocysteine concentration was significantly decreased after 3 months of taking Ocufolin™ [43]. In that study, retinal blood flow measured using Doppler OCT did not reach a significant level. However, only 1 capsule of Ocufolin™ was taken during that study period, which may have been too low a dose to affect retinal blood flow [43]. In contrast, all cases reported here took 3 capsules daily. Case 8 in our study had increased retinal blood flow velocity, which was directly measured using RFI. We hypothesize that decreased homocysteine resulted in improved retinal blood flow due to the mitigation of homocysteine-induced narrowing of retinal Retinal blood flow velocity of the eighth case. Retinal blood flow velocity was measured during study period of 6 months when the patient took Ocufolin™ artery caliber [44]. Since Eyefolate™ and Ocufolin™ lower homocysteine [43], their use seems to lead to an improvement in retinal blood flow, resulting in the improvement of retinopathy. The findings of our series of patients with retinopathy support the speculation, although further clinical studies are necessary.
Several vascular diseases such as ischemic cerebral stroke, small vessel disease, and retinal vein occlusion have been found to associate with MTHFR polymorphisms [28,37,45]. Also, MTHFR polymorphisms are commonly recognized risk factors for retinal vascular disease, including DR progression [34]. Case 5 seems to demonstrate improvement in retinal vein occlusion after the use of the medical food. Huo et al. reported that folic acid therapy could reduce the risk of the first stroke, suggesting that a similar approach may slow the progression of vascular disease [8]. Studies show that Lmethylfolate is a better choice than folic acid with reduced toxicity and better penetration of the central nervous system and retina [30][31][32].
The improvement of DR of our patients that may also in part be attributed to the pharmacological doses of vitamin D in the present study. Vitamin D plays a role in anti-inflammatory and immunosuppressive activities and its potential inhibitory effect on angio-neogenesis has been hypothesized [24,46,47]. Hypovitaminosis of vitamin D increases the risk of DM [46][47][48]. Among DM patients, vitamin D deficiency is found to associate with a higher risk of DR [24,[46][47][48]. The potential relationship between vitamin D status and the DR severity is still controversial [49,50]. Brown reported a case of branch retinal artery occlusion treated more than 24 h post occlusion with Ocufolin™ with a remarkably improved outcome [29]. Our findings support these earlier reports that the microvascular abnormalities (i.e., hemorrhages, MAs, exudates, and macular edema) were reduced after taking the products containing Lmethylfolate and vitamin D.
BCVA is an important component of the visual function. Stability or improved BCVA is the desired outcome treatment of DR. However, DR is often associated with other vision damaging diseases. Case 2 showed a decreased BCVA, which may be due to concurrent AMD. In other cases, amblyopia, cataract, and preexisting vascular disease affected the visual outcomes. Encouragingly, the BCVA in the majority of cases remained stable during the study period. Further study with larger sample sizes and more strict inclusion criteria may help identify the effect of these nutrients on BCVA.
As the first report of a retrospective series of cases that shows a beneficial effect of nutritional supplementation of vitamin D and L-methylfolate on DR and HR, our study also has several limitations. It is not prospective. The lack of a quantitative tool impairs objective observation of the progression of DR and quantification of the dose effect. Further prospective studies with more advanced image modalities such as OCT angiography may provide a better understanding of the benefits to the retinal microvasculature. Second, all patients identified with this positive response were adult Caucasians. Prospective studies with larger sample sizes and community-based studies can stratify patients with racial diversity and their outcome. Third, as mentioned above, the other ophthalmic conditions of these subjects may affect the observations, due to the limited case series. Further prospective large-scale and double-blind studies are needed. Fourth, although the retinal blood flow was increased in case 8, results from a larger sample size are needed to validate the findings. Our ongoing prospective retinal blood flow trial with Ocufolin™ intervention will shed light on the mechanism of these unusual and encouraging case reports, as reported in the present study.
In conclusion, we report a series of DR and HR cases with MTHFR polymorphisms and improvement of retinal microvasculature (mainly hemorrhages, MAs, and exudates) in fundus photographs. It appeared that the use of a carefully formulated medical food which includes L-methylfolate and vitamin D may be effective in facilitating the improvement of DR. Future clinical trials are also needed to show optimal dosing for such medical foods and supplements.