Effect of an oral supplementation with a formula containing R-lipoic acid in glaucoma patients

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ARCHIVOS DE LA SOCIEDAD ESPAÑOLA DE OFTALMOLOGÍA www.elsevier.es/oftalmologia

Original article

Effect of an oral supplementation with a formula containing R-lipoic acid in glaucoma patients夽 S.M. Sanz-González a,b , J. Raga-Cervera a,c , M. Aguirre Lipperheide d , V. Zanón-Moreno a,b,e,i , V. Chiner f , A.I. Ramírez b,g,∗ , M.D. Pinazo-Durán a,b,h,∗ a

Unidad de Investigación Oftalmológica Santiago Grisolía/FISABIO y Grupo de Investigación de la Universidad de Valencia en Oftalmo-biología Celular y Molecular, Valencia, Spain b Red de Oftalmología de la RETICS: RD16-0008: «Prevención, detección precoz, tratamiento y rehabilitación de las patologías oculares», Instituto de Salud Carlos III, Madrid, Spain c Departamento de Oftalmología, Hospital de Manises, Manises, Valencia, Spain d Nua Biological Innovations S.L., Erandio, Vizcaya, Spain e Universidad Internacional de Valencia, Área de Salud, Valencia, Spain f Departamento de Oftalmología, Hospital Universitario Dr. Peset, Valencia, Spain g Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Universidad Complutense de Madrid, Madrid, Spain h Departamento de Cirugía, Facultad de Medicina y Odontología, Universidad de Valencia, Valencia, Spain i Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina y Odontología, Universidad de Valencia, Valencia, Spain

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective: To analyse the safety and effectiveness of the oral administration of a commer-

Received 1 September 2019

cialised supplement containing R-alpha lipoic acid (ALA), taurine, vitamins C and E, lutein,

Accepted 19 November 2019

zeaxanthin, zinc, copper and docosahexaenoic acid (DHA), in patients with primary open

Available online xxx

angle glaucoma (POAG), and in control subjects.

Keywords: Glaucoma

ing 30 participants of both genders that were divided into: POAG Group (n = 15) and a control group (CG; n = 15), assigned to the oral intake of NuaDHA preparations Vision®

Dry eye

(1 pill/day) + NuaDHA 1000 (2 pills/day) for 6 months. Participants were interviewed, oph-

Material and methods: A prospective study of cases and controls was carried out, includ-

Oxidative stress

thalmologically examined, and peripheral blood was taken for routine analysis and the

Nutritional supplements

determination of the pro-oxidant [malondialdehyde (MDA)] and total antioxidant status

Polyunsaturated fatty acids

(TAS). Statistical analysis was performed using the SPSS 22.0 program.

R-lipoic acid

Results: After 6 months of supplementation, there was a significant increase in the plasma TAS (1.073 ± 0.090 mM vs 1.276 ± 0.107 mM, P = .028), along with a parallel decrease in MDA (7.066 ± 1.070 ␮M vs 2.771 ± 0.462 ␮M, P = .005) in the POAG group. The MDA also decreased in the control group (6.17 ± 1.336 vs. 2.51 ± 0.391, P = .028). The Schirmer test improved (20–30%) and the subjective dry eye signs/symptoms noticeably decreased in the POAG group versus the CG.

夽 Please cite this article as: Sanz-González SM, Raga-Cervera J, Aguirre Lipperheide M, Zanón-Moreno V, Chiner V, Ramírez AI, et al. Efecto de la suplementación oral con una fórmula que contiene ácido R-lipoico en pacientes con glaucoma. Arch Soc Esp Oftalmol. 2020. https://doi.org/10.1016/j.oftal.2019.11.009 ∗ Corresponding authors. E-mail addresses: [email protected] (A.I. Ramírez), [email protected] (M.D. Pinazo-Durán). ˜ ˜ S.L.U. All rights reserved. 2173-5794/© 2019 Sociedad Espanola de Oftalmolog´ıa. Published by Elsevier Espana,

OFTALE-1601; No. of Pages 10

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Conclusions: Formulations containing antioxidant vitamins, ALA and DHA, administered for 6 consecutive months, counteracted the oxidative stress by further stabilising the morphological/functional parameters of both the ocular surface and the glaucoma, without presenting with adverse effects or intolerances. ˜ ˜ S.L.U. All rights © 2019 Sociedad Espanola de Oftalmolog´ıa. Published by Elsevier Espana, reserved.

Efecto de la suplementación oral con una fórmula que contiene ácido R-lipoico en pacientes con glaucoma r e s u m e n Palabras clave:

Objetivo: Analizar la seguridad y efectividad de la administración oral de un suplemento

Glaucoma

comercializado que contiene ácido alfa-R-Lipoico (ALA), taurina, vitaminas C y E, luteína,

Ojo seco

zeaxantina, zinc, cobre y ácido docosahexaenoico (DHA) en pacientes con glaucoma pri-

Estrés oxidativo

mario de ángulo abierto (GPAA) y sujetos control.

Suplementos nutricionales

Material y Métodos: Se realizó un estudio prospectivo de casos y controles que incluyó 30

Ácidos grasos poliinsaturados

participantes de ambos sexos, divididos en: pacientes con GPAA (GGPAA; n = 15) y sujetos sanos (GC; n = 15) asignados a tomar durante 6 meses los preparados NuaDHA Visión® (1

Ácido R-lipoico

comp./dia) + NuaDHA 1000 (2 comps./dia). Los participantes fueron entrevistados, examinados oftalmológicamente, extrayendo sangre periférica que fue procesada para analítica convencional y determinación de actividades pro-oxidante [malonildialdehido (MDA)] y estado antioxidante total (EAT). El análisis estadístico se realizó mediante el programa SPSS 22.0. Resultados: Tras 6 meses de suplementación los niveles plasmáticos de EAT aumentaron significativamente 1,073 ± 0,090 mM vs 1276 ± 0,107 mM, p = 0,028, disminuyendo en paralelo los de MDA 7,066 ± 1,070 ␮M vs 2761 ± 0,462 ␮M, p = 0.005 en el GGPAA. El MDA también descendió significativamente en el GC 6,17 ± 1336 vs 2,51 ± 0,391, p = 0,028. Los pacientes con GPAA mostraron valores del test de Schirmer notablemente mayores 20-30%) y mejoraron subjetivamente los signos/síntomas de ojo seco, frente a los resultados del GC. Conclusiones: Las formulaciones que contienen vitaminas antioxidantes, ALA y DHA administradas durante 6 meses consecutivos contrarrestaron el estrés oxidativo, y estabilizaron los parámetros morfológicos/funcionales de la superficie ocular y del glaucoma, sin presentar efectos adversos o intolerancias. ˜ ˜ S.L.U. Todos de Oftalmolog´ıa. Publicado por Elsevier Espana, © 2019 Sociedad Espanola los derechos reservados.

Introduction Glaucoma is a polygenic inheritance disease having ocular hypertension as main risk factor which leads to the death due to apoptosis of retinal ganglion cells (RGC) and optic nerve fibers, giving rise to degenerative and irreversible optic neuropathy that ends up in loss of vision.1 As indicated by the International Council of Ophthalmology (ICO), «in Western countries loss of vision due to open angle glaucoma is more frequent in comparison with Eastern Asia, where loss of vision due to closed angle glaucoma is more frequent».2 It is estimated that 50% of subjects with glaucoma remain undiagnosed. The main risk factor is intraocular pressure (IOP), although familial glaucoma history, aging, diabetes mellitus, myopia or some types of uveitis or cataracts should also be taken into account. The only currently available therapeutic action is hypotensor treatment which successfully reduces

IOP in the majority of patients but does not halt glaucomatous neurodegeneration.3,4 Taking into account the importance of genetic factors,5 some of the pathogenic mechanisms of primary open angle glaucoma (POAG) include oxidative/nitrosative stress with the formation of reactive species and macromolecule damage,6,7 alteration of retrograde axoplasmatic flow and deprivation of essential trophic factors, as well as diminished optic nerve perfusion, giving rise to vascular compromise and ischemic processes.8 The first studies on diet and ophthalmological diseases were carried out in the 19th and later on in the 20th century with the discovery of vitamin A, researching alterations related to lack of vitamins. However, despite large population studies and multiple experimental studies, establishing a cause-effect relationship in the development or progression of ocular diseases remains a controversial issue, mainly due to the diversity of involved factors, differential characteristics

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thereof and the design range of said studies. The majority of studies have focused on the effects of supplements with antioxidants and omega-3 fatty acids (␻-3; docosahexaenoic acid: DHA) on cataracts, age-related macular degeneration, diabetic retinopathy and glaucoma,9 the latter to a lesser extent. According to Granado et al.10 there are no recommendations on specific dosage for many diet components, i.e., carotenoids or flavonoids, that probably (not definitively) produce beneficial results for the body. There is insufficient evidence to recommend increased consumption of fruits and vegetables,11 mainly for the population at higher risk of developing said pathologies. DHA is an ␻-3 essential fatty acid with cytoprotective and cytotherapeutic properties that regulates numerous metabolic processes. At the visual level, DHA has been related to the integrity of the ocular surface and the retina. Several authors have agreed that plasmatic/tissue levels of ␻-3 fatty acids are diminished in patients with glaucoma.12 When these patients take DHA and vitamin E and B supplements they exhibit significant visual field differences when compared to patients who were not administered said supplements.13 However, even though oxidative stress increase has been described in the course of glaucoma,6 other studies of the authors’ research group that analyzed supplementation with antioxidants and ␻-3 fatty acids did not find differences in structural and functional outcomes in patients who were administered said supplements when compared to those who did not take them.14 R-lipoic acid (ALA) is a relatively new nutrient that has acquired relevance in the study of glaucoma.15 ALA is a short chain fatty acid with potent antioxidant and antiinflammatory actions as well as platelet anti-aggregation properties that protects against hypoxia/reperfusion. Its action is probably based on the suppression of apoptosis and autophagia.16 Positive effects have been described after the oral administration of ALA in several pathologies including cardiovascular disease, diabetes and retinal ischemia.17–20 Despite scientific evidence supporting the involvement of oxidative stress in the pathogenesis of POAG, interventionist studies with nutraceuticals have given rise to a scientific debate that subsists to date. Accordingly, we have designed an open case and control study with a six-month supplementation of 2 nutritional complexes with antioxidants: NuaDHA Visión® (Laboratorios Nua Biological Innovations, Erandio, Bizkaia, Spain) + supplementation with DHA: NuaDHA® 1000. in patients with slight-moderate POAG by means of clinical and biochemical assessment.

Material and methods A prospective, interventionist, open case and control study to assess the effects of oral supplementation with NuaDHA Visión® + NuaDHA® 1000 during 6 months. Table 1 summarizes the detailed formulation of said supplements. All the tests and procedures of the study were carried out in accordance with the Helsinki declaration for experimentation with humans and complied with regulations in force in

Table 1 – Formulation and daily dosage of nutrients supplied in the intake of NuaDHA Visión® reinforced with NuaDHA® 1000. Ingredients

Daily supplied dose (mg)

Vitamin C Vitamin E Lutein Zeaxhantin Zinc Copper ␣-R-Lipoic acid Taurin ␻-3 fatty acids Among which. DHA

180 27.9 10 2 15 1 100 150 2.240 2.000

DHA: docosahexanoic acid; mg: milligrams; ␻-3: omega-3.

the European Union (European Investigation Space). In addition, the study obtained the approval of the Committee on Research with Medicaments Ethics of the Dr. Peset University Hospital (Generalitat Valenciana). The participants were previously informed about the particularities and details of the study and were given a document before signing the appropriate informed consent.

Patients In accordance with the inclusion/exclusion criteria of the study, 30 participants were selected (both male and female and between 40–75 years of age) at the Ophthalmology Department of the Dr. Peset University Hospital and consultation rooms at the Monteolivete Specialty Center in Valencia and were distributed in 2 groups, i.e. the control group (CG; n = 15) and the group with POAG (n = 15) in the initial or slight-moderate phase. POAG diagnostic was based on IOP (Goldmann applanation >21 mmHg, central corneal thickness (>500 microns), and morphological/functional tests (pathological visual field [<-6 dB] and ocular fundus examination [papillar cup over 4/10]). The inclusion criteria for the group of patients with POAG comprised having POAG in the initial or slight-moderate phase and voluntarily agreeing to participate in the study. The CG comprised healthy participants free of any type of glaucoma. The exclusion criteria for participants were absence of any chronic ocular disease, not having undergone surgery or laser in the 6 months prior to beginning the study or having neurodegenerative, chronic or systemic diseases, or suffering cachexia, in addition to not taking oral or topical vitamin supplements other than those provided during the study. Baseline interview and examination was carried out before beginning oral supplementation and at follow-up month 6 (baseline and final visit). Both groups took the supplements described above as per the provided instructions during said period.

Operational definition 1 Personal interview.- Registration of personal/familial and anthropometric data, lifestyle characteristics (work, leisure, physical activity), toxic habits (tobacco, alcohol), ocular

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disease (onset, treatment), comorbidities (diabetes mellitus, arterial hypertension, cardiovascular disease), familial antecedents and other relevant comments. 2 Systematic ophthalmological examination.- This examination determined best corrected LogMar visual acuity, anterior and middle segment biomicroscopy, IOP, papillary study in ocular fundus examination, gonioscopy, visual field examination and optic papilla examination with spectral domain optical coherence tomography. In addition, for the specific analysis of the ganglion cell density (␮m) in POAG the Ganglion cell analysis module was utilized. This module is included in the Cirrus-ii optical coherence device by Carl Zeiss Meditec (Madrid, Spain). For the specific ocular surface assessments, the Ocular Surface Disease Index (OSDI) questionnaire was used as well as qualitative biomicroscopic assessment of the cornea, conjunctival and eyelids and tier quantification with the Schirmer test. 3 Blood extraction.- Blood was taken from the antecubital vein on an empty stomach (08:00 h) for standard analysis (baseline glycemia, glycosylated hemoglobin, total cholesterol and fractions [HDL, LDL], triglycerides, urea, creatinin, total proteins and CDK-EPI, and uric acid). The blood samples collected in tubes with EDTA was centrifuged (2400 rpm, 10 min, 4 ◦ C) to obtain plasma (within 2 h from extraction) that was frozen at −80 ◦ C in eppendorfs for processing and assessing oxidative stress molecules, i.e. malondialdehyde (MDA) and products reacting with thiobarbituric acid (MDA/TBARS),21 and the total antioxidant capacity (TAC),22 assessed with the Cayman kit (Cayman Chemical Company, Michigan, USA) based on the capacity of sample antioxidants to inhibit ABTS® oxidation (2,2’-azine-di[3-ehtylbenzothiazoline sulfonate]) to ABTS® in the presence of metamyoglobin. Under reaction conditions, sample antioxidants produce a reduction of A4450nm, proportional to its concentration in which the antioxidant capacity of the sample for preventing ABTS® oxidation is compared to that of a tocopherol analog (Trolox), and is assessed as mM equivalents thereof. 4 Opinion survey about the nutritional supplement.- The participants were asked for their opinion about the received oral supplements. For each question, the sum of affirmatively responded items, divided by the number of participants per group (sample) multiplied by 100 shows the percentage of subjects who responded the questions.

Table 2 – Lifestyle and habits parameters. POAG (%) Physical exercise 33.3 None 21.4 Slight 28.6 Moderate 21.4 Intense Consumption of tobacco 0 Yes 100 No Alcohol ingestion 78.6 Yes 21.4 No

Control (%)

p

0 33.4 66.7 0

0.095

33.3 66.7

0.015

16.7 83.3

0.003

POAG: primary open angle glaucoma. Percentage (%) of participants. Differences considered to be statistically significant if p < 0.05.

Results Evaluation of demographic and anthropometric parameters and lifestyle habits of participants The demographic characteristics of study participants showed a mean age of 64.27 ± 2.28 years for patients with POAG and 5 2.73 ± 3.92 years for the CG. The mean duration of POAG was a 12.44 ± 2.59 years. The percentage of healthy study participants was 55.6% against 44.4% of patients with POAG. Distribution per sex was a 53.8% males and 46.2% females. The assessed anthropometric parameters, i.e., height, weight and body mass index, did not show significant differences between groups. For the CG, mean height was 165.4 ± 3.1 cm, mean weight was 75.5 ± 4.7 kg and the body mass index was 27.5 ± 1.3; and for the POAG group, the mean height was 163.9 ± 2.7 cm, mean weight was 73.4 ± 4.4 kg and the body mass index was 27.3 ± 1.5. Data on habits and lifestyle of study participants is better illustrated in percentages as shown in Table 2. In addition, the percentage of desertion due to symptomatology related to product intake was minimum: only 10% of all participants (all in the POAG group) reported that they were giving up the supplements, with the main causes being slight-moderate gastroenterological discomfort and swallowing difficulties.

Evaluation of biochemical parameters Statistical analysis The data were statistically analyzed with SPSS 22.0 (IBM Corporation 2012, Armonk, NY, USA). The results presented herein are mean ± standard error. The differences between the results obtained after and before supplementation in each group were studied with the Wilcoxon test. The differences between cases and controls were analyzed at baseline and at the end of the study with the U Mann-Whitney test. For categorical variables related to lifestyle, the Chi square test was used. Differences were regarded as statistically significant with a p ≤ 0.05.

Table 3 shows the biochemical results of change from the blood of participants with the baseline and final values for each parameter as well as the observed variation percentage between the 2 points of the study. When comparing both study groups, statistically significant differences were observed in the following parameters: 1) baseline glucose (p = 0.031), although with values below 120 mg/dL, for which reason they are not clinically relevant for the present study; 2) total protein, with baseline (p = 0.009) and final values (p = 0.048), with variations between groups at baseline and termination of the study below 0.5 g/dL, which did not reach clinic relevance.

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Table 3 – Comparison of biochemical parameters in blood of participants at baseline and of oral supplementation. POAG

Glucose (mg/dL) Urea (mg/dL) Creatinine (mg/dL) FGe (CKD-EPI) Uric acid (mg/dL) CH (mg/dL) HDL (mg/dL) LDL (mg/dL) VLDL (mg/dL) TG (mg/dL) ALT (GPT) UI/L T proteins (g/dL) HbA1c (%) HbA1c (mmol/mol)

CG

Baseline

Final

%

p

Baseline

Final

%

p

100.50 ± 2.88 38.00 ± 2.47 0.84 ± 0.04 81.70 ± 2.04 5.12 ± 0.19 217.40 ± 13.22 57.50 ± 4.90 136.40 ± 12.70 23.50 ± 2.96 117.60 ± 14.69 22.50 ± 4.07 6.82 ± 0.12 5.33 ± 0.08 34.70 ± 0.99

93.30 ± 2.34 41.10 ± 2.92 0.83 ± 0.04 82.40 ± 2.39 5.30 ± 0.23 214.60 ± 16.35 54.40 ± 5.69 139.90 ± 15.34 20.30 ± 2.42 101.70 ± 1.21 19.70 ± 3.75 7.03 ± 0.12 5.43 ± 0.08 35.90 ± 0.89

−7.16 8.16 −0.84 −0.36 3.52 −1.29 −5.39 2.57 −13.62 −13.52 −12.44 3.08 1.88 3.46

0.019 0.154 0.475 0.878 0.067 0.284 0.138 0.646 0.137 0.139 0.057 0.042 0.023 0.024

89.86 ± 3.21 38.88 ± 3.66 0.88 ± 0.05 91.36 ± 4.54 5.05 ± 0.34 193.57 ± 9.84 53.64 ± 3.18 120.21 ± 8.04 18.17 ± 2.61 99.29 ± 12.36 22.29 ± 2.24 7.29 ± 0.12 5.42 ± 0.07 34.67 ± 1.43

98.67 ± 2.31 37.80 ± 1.62 1.58 ± 0.62 83.00 ± 3.29 5.86 ± 0.28 198.27 ± 8.75 49.33 ± 2.68 131.13 ± 7.50 17.80 ± 1.44 88.33 ± 7.27 25.73 ± 3.79 7.35 ± 0.08 5.40 ± 0.09 35.50 ± 0.97

9.80 −2.77 78.37 −9.15 16.04 2.43 −8.03 9.08 −2.02 −11.03 15.47 0.85 −0.40 2.40

0.048 0.726 0.055 0.462 0.133 0.005 0.048 0.752 0.330 0.972 0.506 0.339 0.180

ALT (GPT): alanin aminotransferase (pyruvic glutamic transaminase); CG: control group; CH: total cholesterol; FGe (CKD-EPI): glomerular filtrate estimated with CKD-EPI formula; POAG: primary open angle glaucoma; HbA1c: glycosylated hemoglobin; HDL: high density lipoprotein cholesterol fraction; LDL: low density lipoprotein cholesterol fraction; T proteins: total proteins; TG: triglicerids; VLDL: very low density lipoprotein cholesterol fraction. Where % = [(final-initial)*100)]/initial. Data represent mean ± standard error. Differences regarded as a statistically significant if p < 0.05 shown in bold type in the table.

Evaluation of oxidative stress markers

Evaluation of ophthalmological parameters

Fig. 1 shows oxidative stress markers data in the period before and after supplementation. In what concerns TAC, values were significantly diminished in patients with POAG (1.073 ± 0.090 mM at baseline. However, the values increased significantly at the end of the followup (1.276 ± 0.107 mM; p = 0.028), an increase of 20% at the end of the supplementation period. In addition, a 14.8% increase was observed in the TAC of the CG group at the end of the supplementation period. Analyzing the MDA levels, the CG showed a reduction between the 2 follow-up analyses (6.167 ± 1.336 ␮M [pre-] and 2.509 ± 0.391 ␮M [post-] [p = 0.028]). In addition, the POAG group showed highly significant reductions between the two points of the study (7.066 ± 1.070 ␮M at 2.761 ± 0.462 ␮M; p = 0.005). Our data demonstrate that plasmatic MDA diminished significantly in both groups who underwent nutritional intervention (61% in the CG and 59% in the POAG group). However, a comparison of both groups did not produce differences in the data taken at baseline (p = 1) and at the end (p = 0.476) of the study.

The ophthalmological examination did not show statistically significant differences in any parameter in the comparison of baseline and end of study data in the POAG group or in the CG. Differences were not observed in the majority of analyzed parameters when comparing case vs. controls both at baseline and at the end of the study. The subjects with POAG showed increased standard deviation which, even though not being statistically significant, reached 9.65% for the right eye (RE; p = 0.500) and 9.80% for the left eye (LE; p = 0.465). In what concerns RGC density, the study data demonstrated that cell density (␮m) was significantly lower in the POAG group than in the CG, both at baseline (p = 0.004) and at study end (p = 0.046). Clinic examination of the ocular surface with biomicroscopy and quantitive examination techniques of the surface lacrimal film demonstrated higher prevalence of dry eye in the POAG group than the CG. Almost 2/3 of the POAG group insisted that they did not have dry eye (or were not aware of the condition) in the baseline interview. Comparing cases versus controls, statistically significant differences were found at baseline (p = 0.001) as well as at the end of the study (p = 0.027). The variations were determined as the amount of tears of each participant at baseline and oral supplementation, observing a positive tendency in this parameter. The percentage of TM volume increase (Schirmer test in mm) reached 20–30% (RE: p = 0.115; LE: p = 0.039) in patients with GGPA, and 5–20% (RE: p = 0.213; LE: p = 0.046) in CG subjects.

Evaluation of the opinion survey on the nutraceutical supplement Table 4 shows the results of the opinion survey on the nutraceutical supplement showing and affirmative response percentages of participants of each group. Quantitatively, patients with POAG responded more in the affirmative to questions 1, 2, 3 and 9 than the CG, which confirms the subjective improvement of the analyzed parameters in what concerns vision together with an improved mood and, as indicated in the responses, harder nails and softer skin.

Discussion In a 6-month follow-up with patients having POAG, oral administration of NuaDHA Vision® containing ALA and reinforced with added DHA (NuaDHA® 1000) exhibited a capacity to counteract plasmatic oxidative stress and improve the

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A

TAC (mM)

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Baseline

Baseline

End

End

CG

POAGG

p= 0.144

p= 0.028

B

MDA (µM)

9 8 7 6 5 4 3 2 1 0

Baseline

End

Baseline

End

CG

POAGG

p= 0.028

p= 0.005

Fig. 1 – Determination of oxidative stress parameters in both groups of participants throughout the study. The results are indicated as mean ± standard deviation. Statistical significance is p ≤ 0.05. TAC: total antioxidant condition; CG: control group; POAGG: primary open angle glaucoma group; MDA: malonyl dialdehyde.

Table 4 – Opinion questionnaire at the end of the oral supplements administration period. In your opinion, the oral supplementation

1.- Has your eyesight improved? 2.- Have you noticed having more energy? 3.- Do you find your mood has improved? 4.- Did you have digestive problems before beginning this study? 5.- Did you experience discomfort due to taking DHA? 6.- Have you noticed increased abilities in your memory? 7.- Was it easy to take the softgels? 8.- Did you take the softgels during dinner? 9.- Do you think that the condition of your skin and nails has improved? 10.- Have you noticed less hair loss?

% POAG

Control

27.27 27.27 27.27 36.36 45.45 9.09 45.45 45.45 36.36 9.09

14.29 0.00 7.14 21.43 28.57 7.14 50.00 64.29 14.29 7.14

DHA: docohexanoic acid; POAG: primary open angle glaucoma; %: percentage of individuals with positive response.

integrity of the ocular surface in patients with chronic administration of hypotensor eyedrops. Supplementation provided a group of nutrients that includes parts of the AREDS 2 formulation (vitamins C, E, lutein, zeaxhantin, zinc and copper) at the maximum concentrations according to European regulations in force, with the

addition of 3 nutrients: taurin, ALA and DHA.23–25 Taurin is the most abundant aminoacid in the retina26 and exhibits a neuroprotective effect over the RGC.24,27,28 DHA, the most abundant polyunsaturated fat in the macula29 is a crucial nutrient for the integrity of the ocular surface30 and for the RGC.31,32 Lastly, ALA has demonstrated additional potential in patients with

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glaucoma to protect the RGC,15,33,34 as well as beneficial effects for the lacrimal film.35 Management of oxidative levels is essential for the adequate function of our body and has become a subject of great interest in biomedical research, to the extent that the 2019 Nobel Medicine award was given to Kaelin, Semenza and Ratcliffe for their discoveries on several mechanisms capable of acting as sensors to identify the presence and availability of oxygen, and on the cellular processes to adapt to the changes inflicted by oxygen. Oxidative stress can also impact the visual system by inducing several pathologies.6,36–40 Oxidative stress36,40,41 is related to the deterioration of the trabecular mesh in patients with POAG, compromising the outflow of the aqueous humor36 and contributing to RGC dysfunction.42 The present study assessed lipidic peroxidation metabolites (MDA/TBARS), and TAC in patients with POAG and healthy subjects before and after oral supplementation with a formulation including R-lipoic acid during a period of 6 months. The baseline MDA/TBARS values were higher in patients with POAG than in the CG, matching reports by other authors.43–45 After 6 months of supplementation, results evidenced a significant reduction in plasmatic oxidation (60% in both groups) together with an increase in antioxidant power which was higher in patients with POAG (20%) against 14.8% in the CG. These results also agree with those of other authors in relation to diabetic retinopathy46,47 or age-related macular degeneration.48 However, no reference was found to preand post-supplementation assessments as those included in the present study. Accordingly, the present results are of interest and confirm the capacity of the essayed formulation to contain the potential damages derived from excessive oxidation.36,37,45 Analyzing the differences for oxidative stress parameters, i.e. TAC and MDA, at baseline and at study end between the CG vs. the POAG group, no significant differences were observed in MDA although the values thereof are lower in the CG than in the POAG group. In what concerns TAC, similar values were observed in both groups both at baseline and at the end of the supplementation. This result differs from those found in the literature,43–45,49 that report an association of higher MDA levels and lower TAC levels in the POAG group. This could be related to the number of participants in the study which could have influenced and diminished the statistical power for the analyzed parameters. The improvement observed in the overall antioxidant activity could be partly explained by means of the formulation (vitamins C, E, zinc, lutein, zeaxhantin and ALA) together with a capacity of DHA to activate endogenous production of glutathione and catalase,50 an effect that is shared with ALA.51 Doubtlessly, the latter is an innovative ingredient in the formulation because its capacity involves deploying antioxidant power in aqueous as well as in fatty media as well as renewing important antioxidants such as vitamins C, E or gluthation. In addition, ALA provides a marked neuroprotective effect because it is able to stimulate the expression of nerve growth factors and support communication of motor nerves. At the level of the RGC, ALA is considered to be an agent capable of containing oxidative stress while supporting mitochondrial function, counteracting factors which tend to generate ganglion cell apoptosis.33,52 In summary, the char-

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acteristics of ALA make it an ideal candidate to control RGC dysfunction and apoptosis as much as possible, for which reason it is of great interest for the management of glaucoma.15 It must be emphasized that the most widely utilized ALA ingredient in supplements is the RS-racemic form. However, the present study utilized the R-form, regarded as more active and bioavailable and therefore the most desirable form to achieve the maximum potential of this nutrient. From the beginning of the study and during the followup period, patients with POAG were treated with hypotensor eyedrops and therefore their IOP was controlled at all times. For this reason it was foreseeable that statistically significant differences between IOP values at baseline and study end would not be found. The rest of analyzed ophthalmological parameters also failed to show significant differences in what concerns oral supplementation and the studied groups during the supplementation period. It is suggested that the six-month supplementation period enabled a stabilization of patients with POAG in what concerns the oral supplementation with the formula that included R-lipoic acid. García-Medina et al.14 assessed ophthalmological parameters before and after supplementation in patients with glaucoma without finding significant differences. The results of the present study related to the ocular surface are worthy of note. Higher prevalence of dry eye was observed in the POAG group even though in the initial interview the patients diagnosed with POAG were not aware of having said disorder. Frequently, the use of hypotensor eyedrops involves alterations of the lacrimal film integrity53 so that many patients with glaucoma exhibit a propensity to subclinical dry eye without becoming aware of the disorder until the symptoms become evident, as it seems to be the case of said study group. Schirmer test results showed a clear tendency to improvement in all participants, even more so in patients with POAG who exhibited tear volume increases of 20–30% (RE/LE) against 5–20% (RE/LE) in the healthy subjects. This positive response could be attributed to the effects of 2 components of the study formulation, DHA and ALA. several authors have documented the benefits of ALA35 and DHA30,54 in the context of the ocular surface. In what concerns DHA, said improvements could be due to the production of a derivate of this fatty acid known as neuroprotectin D1, which has anti-inflammatory and cornea regeneration functions.55 In a study with dry eye patients, Walter et al.56 observed a high ratio of ␻-6:␻-3 fats in tears, which supports the importance of ␻-3 for the ocular surface. The results of the present study confirm that the micronutritional supplement (NuaDHA Vision® and additional DHA) qualitatively and quantitatively supports surface lacrimal film in patients with POAG. Accordingly, this study suggests that said supplement could protect these patients against the dry eye syndrome caused by chronic administration of hypotensor eyedrops with aggressive excipients. Lastly, the opinion survey on the oral supplement used during 6 months showed a majority of positive responses in what concerns improvement at the visual level, in mood and the optimization of the skin and adnexa when compared to the responses given by the healthy subjects. In what concerns analyzed blood parameters, glucose levels on an empty stomach exhibited a significant reduction in

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the POAG group (p = 0.019) after oral supplementation with the formulation of the study during 6 consecutive months. In what concerns glucose, despite the difference between groups, the values are below 120 mg/dL, which means that the subjects are not diabetic; besides, this statistically significant difference lacks relevance in the present study. Even so it is an interesting data because high glucose levels sustained in time could give rise to the formation of “advanced glycation end products” or in other words the adhesion of glucose molecules to important biomolecules such as proteins and fats, in a process that is involved in the aging and dysfunction of multiple tissues.57,58 In the case of glaucoma, said molecules could contribute to increase the stiffness of the cribriform plate.58 The advanced glycation end products are particularly abundant in diabetic patients and therefore the improvement observed in glycemia levels of patients with glaucoma could also be relevant for patients with diabetic retinopathy as it will assist in containing damages derived from an excessive glycemic burden which, together with the reduction of observed oxidative stress, could doubtlessly contribute to the improvement of said disorder. The observed hypoglycemic effect that contributes to contain the generation of advanced glycation end products is mainly due to the ALA included in the formulation.59,60 On the other hand, variation in total protein numbers between groups, both at baseline and at study end, is below 0.5 g/dL. In addition, the observed tendency is similar in both groups of patients and in both time periods, for which reason the clinic relevance of this data is very low considering that the authors have not found studies on this subject carried out under similar conditions. To end, the limitations of the present study include: 1) the small number of participants although it must be taken into account that this is a pilot study and that the authors aim to continue increasing the sample size; 2) it should also be considered that the follow-up period is relatively short, and this will be taken into account for new studies with longer supplementation periods to improve statistical power; 3) in addition, dietary variabilities between patients were not controlled. This is an important factor because it probably influenced the end results, and therefore diets must be taken into account in subsequent studies of this type. However, during the study it was taken into account that in the months prior to initiating oral supplementation the patients were requested not to take any other micronutritional supplement that included antioxidants and DHA, as explained above. To conclude, oral supplementation during 6 consecutive months with 2 simultaneous formulations (NuaDHA Vision® + NuaDHA1000® ) produced an increase of antioxidant defenses and a reduction of pro-oxidative activity in the plasma of study participants. Therefore, it is suggested that said supplementation could be useful as adjuvant for chronic treatment of patients with POAG, which also improved ocular surface integrity in said patients.

Conflict of interests No conflict of interests has been declared by the authors of this paper. Mercedes Aguirre Lipperheide is an executive of Nua Biological Innovations, S.L.

Acknowledgments This paper is partly a cooperation between research team members of the Ocular Pathology Ophthalmology Research Network (OFTARED) in Madrid and Valencia, identified with PN I+D+i 2016-2020; Silvia M. Sanz-González has been employed by FISABIO by means of a researched donation provided by Nua Biological Innovations, S.L. for the development of this project.

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