Blueberry Effects on Dark Vision and Recovery ... - ACS Publications

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Blueberry Effects on Dark Vision and Recovery after Photobleaching: Placebo-Controlled Crossover Studies Wilhelmina Kalt,*,† Jane E. McDonald,† Sherry A. E. Fillmore,† and Francois Tremblay§ †

Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, Nova Scotia B4N 1J5, Canada § Department of Ophthalmology and Visual Sciences and Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3G 3G9, Canada ABSTRACT: Clinical evidence for anthocyanin benefits in night vision is controversial. This paper presents two human trials investigating blueberry anthocyanin effects on dark adaptation, functional night vision, and vision recovery after retinal photobleaching. One trial, S2 (n = 72), employed a 3 week intervention and a 3 week washout, two anthocyanin doses (271 and 7.11 mg cyanidin 3-glucoside equivalents (C3g eq)), and placebo. The other trial, L1 (n = 59), employed a 12 week intervention and an 8 week washout and tested one dose (346 mg C3g eq) and placebo. In both S2 and L1 neither dark adaptation nor night vision was improved by anthocyanin intake. However, in both trials anthocyanin consumption hastened the recovery of visual acuity after photobleaching. In S2 both anthocyanin doses were effective (P = 0.014), and in L1 recovery was improved at 8 weeks (P = 0.027) and 12 weeks (P = 0.030). Although photobleaching recovery was hastened by anthocyanins, it is not known whether this improvement would have an impact on everyday vision. KEYWORDS: anthocyanin, blueberry, retina, night vision, clinical

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INTRODUCTION Among the numerous health-promoting properties attributed to blueberries, one of the best recognized is their purported benefits to vision,1 owing to the berry’s high concentration of anthocyanins. The link between blueberries and vision improvements originates in part from a body of research published between the 1960s and 1980s on the effects of in vitro, in vivo, and clinical interventions with the European “blueberry”, the bilberry (Vaccinium myrtillus L.), on various vision parameters (for reviews, see Kalt et al.1 and Canter and Ernst2). For discussion, “blueberries” include those commercially available Vaccinia fruit that have a high anthocyanin (ANC) concentration, including bilberries, wild lowbush (Vaccinium angustifolium Aiton), cultivated highbush (Vaccinium corymbosum L.), and rabbiteye blueberries (Vaccinium asheii Reade). Other berry crops with a high ANC concentration (e.g., black currants) have also been studied in relation to vision (see, for example, refs 3 and 4). Unfortunately, most of the early European clinical research that reported effects of bilberries on night (scotopic) vision did not employ a randomized, placebo-controlled study design and, instead, used a simple prepost design for psychophysical measurements related to vision. Canter and Ernst2 identified 30 clinical trials that related specifically to bilberries and night vision. Of these, 12 trials were placebo-controlled, and of these, 5 trials used a randomized design. Four trials5−8 of the five placebo-controlled randomized trials reported a negative outcome. The fifth trial9 and seven nonrandomized trials10−16 reported positive effects in relation to measures of visual performance in the dark. Canter and Ernst2 cite weak trial designs, a wide range of bilberry products and doses employed, diverse study populations, and differing intervention designs © XXXX American Chemical Society

and conclude that there is currently little evidence to support the claim of night vision improvements due to bilberries. They encouraged more clinical research employing rigorous study designs, modern methods for scotopic vision testing, and more control of intervention factors such as subject age, product form, dose, and study duration. After the 2004 publication by Canter and Ernst,2 the results of a randomized double-blind placebo-controlled study were published17 that examined the vision of 59 volunteers who had consumed twice daily for 4 weeks either specifically fermented grape anthocyanosides or a placebo. In this study an improvement in an objective measure of scotopic contrast sensitivity was reported as well as an improvement in subjective assessments of clinical visual symptoms (e.g., eye fatigue, strain, dryness). Although conclusive clinical evidence may be lacking, in vitro studies show that ANC can stimulate the regeneration of rhodopsin4 and modulate retinal enzyme activity.18 These in vitro results complement in vivo results obtained from electroretinogram studies in animals showing that ANC intervention does affect vision physiology by protecting the retina from photostress damage.19,20 These in vivo effects are also suggestive of ANC effects on retinal physiology. ANCs can become localized in eye tissues of rats, rabbits,21 and pigs.1 ANCs administered via the peritoneum were differentially distributed after 1 h among seven ocular tissues of rats and found to be highest in the sclera and choroid, where it exceeded the plasma ANC concentration by about 100 Received: August 6, 2014 Revised: October 16, 2014 Accepted: October 22, 2014

A

dx.doi.org/10.1021/jf503689c | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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times.21 Several ANCs could be detected in the whole eyeball of pigs1 fed 2.4−12 mg ANC/kg body weight in blueberries for up to 8 weeks and where the concentration in the whole eye appeared to be correlated with blueberry dose. Human intake of ANC is estimated at approximately 200 mg daily in the North American population22 or approximately 2.7 mg/kg body weight for a 72 kg person. Despite the volume of research surrounding ANC effects on vision and their ability to become localized in ocular tissues, there is still a paucity of solid clinical evidence to support existing commercial claims of improvement in night vision to normal-sighted individuals associated with the consumption of blueberries and other ANC-containing foods. This paper describes the results of two placebo-controlled, randomized crossover studies that tested a normal-sighted middle-aged human population for blueberry ANC effects on dark adaptation, scotopic vision, and recovery from photobleaching of the retina.

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Table 1. Summary of Two Clinical Trials, S2 and L1, That Examine Blueberry Anthocyanin Effects on Dark Vision clinical trial short trial with two blueberry products (S2) study design

randomized, double-blind

double crossover

single crossover

vision testing

sites

2

2

testers

2

1

after products

3 weeks

8 weeks 12 weeks

after washout

3 weeks

4 weeks 8 weeks

placebo

red beet powder, 3 capsules

placebo juice (300 mL)

blueberry juice other products

300 mL containing 271 mg C3g eqa blueberry powder, 3 capsules, 7.11 mg C3g eq

300 mL containing 346 mg C3g eq none

no. of subjects

72

59

age and gender

age 35−47 years; M = 11; F = 16 age 48−55 years; M = 9; F = 17 age 56−65 years; M = 7; F = 12 0

age 35−50 years; M = 8; F = 14 age 51−65 years; M = 14; F = 24

vision testing schedule

test products and daily dose

MATERIALS AND METHODS

Study Design and Participants. Two prospective, placebocontrolled, double-blind, crossover studies, S2 (short duration, two products) and L1 (long duration, one product), were conducted to examine human visual function after intake of blueberry products. Vision testing was conducted under reduced light at specific intervals after the consumption of blueberry products, placebo, or nothing (washout). In S2 the assignment of the 72 subjects to two crossover groups was balanced between testing sites (2), gender (2) and age (3) (Table 1). Similarly, in L1 the assignment of the 59 subjects to two crossover groups was balanced between testing sites (2), gender (2), and age (2) (Table 1). There were no drop-outs from S2, whereas in L1 there were two. Partial results of drop-outs were removed from the L1 analysis. Both studies were approved by the research ethics review board of Dalhousie University (Canada) and were in accordance with the tenets of the Declaration of Helsinki. The Clinicaltrials.gov identifier for both S2 and L1 is NCT01942746. One hundred and twenty individuals were recruited using printed advertisements posted in two rural communities of Nova Scotia, Canada. Inclusion criteria were male or female, age 35−65 years, with no ocular history other than refractive glasses. The minimum criteria for visual function required for inclusion were (1) visual acuity better than 6/7.5 on EDTRS acuity chart (VectorVision, Greenville, OH, USA) at 2.5 m, (2) contrast sensitivity within normal range to all spatial frequencies tested on Visteck 3000 (VectorVision) at 2.5 m, (3) stereoacuity better than 80 s of arc on Frisby stereoacuity test (Clement Clarke International Ltd., Essex, UK), and (4) intraocular pressure lower than 21 mmHg from the average of three measures using a Mentor tonopen-XL (Innova Medical Ophthalmics, Toronto, Canada). In the process of eliminating amblyopia (dimness in vision), candidates were examined for manifest strabismus (unable to focus both eyes on one spot) and anisotropia (nonuniform responsiveness between both eyes). Ophthalmologic examination also included direct retinoscopic assessment of retinal pigment epithelium and retina as well as a slit lamp examination of anterior segment. Family and medical history excluded subjects with family history of retinal degeneration, glaucoma, diabetes, hypertension, cataract, and amblyopia. On the basis of these criteria, 72 and 59 subjects were recruited for S2 and L1, respectively, and were assigned to groups that were age and gender-matched (Table 1). All 59 participants of L1 had previously participated in S2. The S2 was completed approximately 7 months before the commencement of L1. During the intervention and the washout period of both S2 and L1, subjects were instructed to abstain from ANC-containing foods and were provided with a list of alternative fruit choices, such as melons, green grapes, kiwi fruit, green-peeled apples. Compliance to the dietary restriction was assessed using a questionnaire. All subjects were

long trial with one blueberry product (L1)

study population

drop out a

2

C3g eq, cyanidin 3 glucoside equivalents.

considered to be well-nourished before and during the study. During the study their diet was modified only by the restriction on intake of nonapproved ANC-containing products. Test Products. In both S2 and L1 blueberry intake was standardized for ANC content, which was consumed in the form of single-strength blueberry juice (BJ) and in S2 also as a freeze-dried BJ powder. BJ was produced using a commercial process (VanDyks Health Products, Caledonia Nova Scotia, www.vandykblueberries.ca) using previously frozen blueberries that were equal proportions of the cultivars ‘Tifblue’ (V. asheii Reade) and ‘Rubel’ (V. corymbosum L.). The composition of these blueberry cultivars has been reported.23 Pasteurized juice was stored in 350 mL clear glass bottles, kept refrigerated, and stored in the dark until delivery to subjects and for no longer than 3 months. In S2 colorimetric analysis of ANCs24 at the time of BJ production indicated an ANC concentration of 6.04 (SD = 0.18) mg of C3g eq/g dry weight. In L1, BJ had an ANC concentration of 6.83 (SD = 0.20) mg C3g eq/g dry weight at the time of production that declined by 19% to 5.52 (SD = 0.09) mg C3g eq/g dry weight by the end of 3 months of refrigerated storage. In both S2 and L1, 300 mL of BJ and in L1 300 mL of placebo juice composed of food grade ingredients (Table 2) were consumed daily. The BJ provided subjects in S2 with a daily ANC dose of 271 mg C3g eq and in L1 with 346 mg C3g eq (Table 1). The BJ powder used in S2 was produced from freeze-dried BJ and packaged in gelatin capsules. Each capsule contained 2.37 mg C3g eq per capsule; three capsules were consumed for a total daily ANC intake of 7.11 C3g eq. In S2 the placebo was freeze-dried red beet powder in gelatin capsules. Red beet color is due to betalain pigments. Red beets do not contain ANCs; their nutrient composition B



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identify the position of the light stimulus. Up to 20 s was allowed for the subject to determine the position of the light stimulus on the monitor (upper or lower portion). This task was repeated once every minute for 30 min. To compensate for the limitations of the monitor’s range of light intensities as subjects became dark adapted, subjects wore neutral density filtered goggles as their DA progressed in both S2 and L1. Goggles were applied when the monitor’s intensity was close to the minimum range, where linearity in luminosity was more variable. In L1 standard retinal photobleaching before DA testing employed a light intensity of 25000 lx for 120 s. In contrast to S2 DA testing, in the L1 the intensity of the dim light was decreased until it could no longer be perceived. Although these two strategies (increasing vs decreasing light intensity) yielded different absolute threshold values between S2 and L1, there was no impact on the DA dynamic changes. Visual Acuity (VA) Testing. To test scotopic VA after the 30 min DA test, subjects continued to wear the light attenuation goggles while single black optotypes of varying size with crowding bars against a white background were presented to them. There were eight different optotypes (letters) of similar discrimination difficulties.26 Optotype light intensity was 100 cd/m2 for the background and 0.316 cd/m2 for the black letters. Optotypes were first shown at a small size and then their size was increased until the subject could correctly identify the letter. The VA values were measured as the minimum size of the optotype that was correctly perceived through a staircase threshold approach with the third to the sixth reversals averaged for a final value. With this method the threshold scotopic VA was measured 12 times per session. Contrast Sensitivity (CS) Testing. While still in darkness and with the subject wearing the light attenuation goggles, sine wave gratings of three spatial frequencies (1, 3, and 10 cycles/degree) were presented at various levels of contrast, starting at 50% of full black/white contrast using the Michaelson contrast equation.27 A staircase method was used to determine the minimum contrast discernible. The three spatial frequencies were randomized, and the subject had to correctly determine the orientation of the grating on the monitor (−30, 0, or 30°). Following a correct answer the tester decreased the contrast, whereas after an incorrect answer, the tester increased the contrast. The mean of eight final reversals was considered as the CS threshold for each of the spatial frequencies. In our statistical analysis, we retained only the CS threshold, expressed in percent contrast, for one of the three spatial frequencies used (3 cycles/degree). Macular Stress Recovery (MSR) Testing. For the MSR test, the light attenuation goggles were removed. Then VA was measured as the smallest readable optotype from a computerized five-line, five-letter chart similar to EDTRS charts with a white background intensity set at 100 cd/m2. After VA was established in those conditions, retinal photobleaching was achieved when one eye was exposed to a diffuse light of 3.5 × 104 cd/m2 (intensity measured at the cornea) for 20 s in S2 or, in L1, to 8.0 × 104 cd/m2 for 20 s. After this bleaching exposure, the subject was presented again with the EDTRS chart, and the time was measured to recover VA to one line above the smallest optotype before retinal photobleaching. To ensure minimal bias, EDTRS charts presented after bleaching were different from those presented before bleaching, however, with letters of the same reading difficulty level. Due to the nature of the bleaching process, the test could be conducted only once during a vision testing session. Statistical Analysis. Experimental Design. Data were analyzed using the ANOVA procedure in GenStat.28 S2 and L1 employed a crossover design that was balanced between testing sites, age, and dietary treatment. In both trials all participants were tested with every dietary treatment and with washout periods between each treatment. In both S2 and L1 all data were evaluated for normality and, when necessary, data were converted to log10 to achieve normal distribution of variation. In S2 the analysis model was product (i.e., placebo, BJ, and BJ capsule) for fixed effects and test site, week, and age for random effects. In L1 the analysis model was product (i.e., BJ and placebo) for fixed effects and week and age for random effects. In both S2 and L1 covariation for each subject’s performance on vision tests before the study began (pretreatment) was identified and corrected. In S2 the

Table 2. Composition of Blueberry Juice Placebo Used in Trial with Long Intervention and One Blueberry Test Product (L1) ingredient

percent (w/w)

water fructose glucose ascorbic acid citric acid sodium citrate malic acid red color blue color cranberry flavor blueberry flavor xanthum gum

88.37 4.0 4.0 0.5 0.5 0.3 0.3 0.70 0.02 0.60 0.70 0.015

total

100.00

has been described.25 Subjects were instructed to consume their test product before noon. Dietary compliance was assessed in L1 using a questionnaire where full compliance for test product consumption was defined as having taken a total 300 mL of BJ or placebo at least 5 days per week. Full compliance to the ANC-free dietary restriction was defined as two or fewer servings of ANC-containing food per week. No adjustment was made to account for missed doses of product or noncompliance to the ANC-free diet. Product Intake and Vision Testing. In S2 vision was tested two times and in L1 vision was tested three times before the diet intervention commenced so that subjects could become accustomed to the way in which the vision tests were conducted. In S2 after this pretreatment testing, subjects consumed BJ products or placebo daily for 3 weeks. Randomization was carried out to assign the order of dietary treatment to each subject. In S2 each subject had their vision tested after 3 weeks of product intake and then again after 3 weeks of washout until all three products (BJ, BJ capsule, placebo) were tested. This 3 week cycle to test three products each took 18 weeks. In L1 after pretreatment testing and randomization, vision was tested after 8 and 12 weeks of daily BJ or placebo intake and after 4 and 8 weeks of washout. In L1 this cycle of two products and two washouts took 40 weeks. Vision was tested at the same time of the day (±2 h) within an individual to avoid possible diurnal effects on vision. No attempt was made to schedule vision testing at a fixed period after consumption of BJ, BJ capsules, or placebos. Vision was always tested by the same person and at the same location (Table 1). A vision testing session lasted 90° between the two trials. The perpendicular line segment distances for the blueberry products were shorter in S2 than in L1, suggesting a closer relationship between CS and blueberry intake in S2, which used a shorter intervention. The effect of blueberry ANC on DA is of great interest to the field, and in this paper DA performance is indicated in the biplot as DAthr, the final dark threshold. In both S2 and L1 DAthr was strongly associated with the first PCA score, because its cosine angle was small in relation to score 1. Score 1 contributed the greatest percentage (63% in S2; 52% in L1) of the variation illustrated by the biplot, indicating that the DAthr was associated with high variation (Figure 4). The length of the perpendicular line segments from the dietary treatments to the DAthr vector varied, suggesting no real pattern in the DAthr response in relation to the diet treatment. VA also contributed strongly to score 1, particularly in L1. VA and DAthr were strongly and negatively correlated.

Table 4. Dark Adaptation Test Results Calculated from Fitted Curves Obtained from Trial (LI) with Long Intervention Period Examining One Blueberry Test Product DACRTa (min) juice placebo grand mean SEM F prob, treatment effect size juice placebo grand mean SEM F prob, treatment effect size juice placebo grand mean SEM F prob, washout effect size juice placebo grand mean SEM F prob, washout effect size

DAAUCb

DAt90%c

DAthrd

(min)

(10-2cd/m2)e

Mean, 8 Week Treatment 6.24 37.90 22.22 6.53 37.61 22.46 6.38 37.76 22.34 0.189 0.556 0.818 ns ns ns 0.20 0.07 0.04 Mean, 16 Week Treatment 6.55 36.66 20.82 6.97 36.34 20.63 6.76 36.50 20.72 0.236 0.485 0.562 ns ns ns 0.23 0.09 0.04 Mean, 4 Week Washout 6.44 37.10 22.23 6.59 37.79 22.22 6.51 36.95 22.22 0.454 0.539 0.871 ns ns ns 0.04 0.20 0.00 Mean, 8 Week Washout 6.50 37.04 21.29 7.21 37.08 20.84 6.85 37.06 21.07 0.526 0.477 0.599 ns ns ns 0.18 0.01 0.08

−1.49 −1.52 −1.50 0.024 ns 0.16 −1.54 −1.54 −1.54 0.024 ns 0.00 −1.53 −1.55 −1.54 0.026 ns 0.10 −1.51 −1.50 −1.51 0.024 ns 0.05

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DISCUSSION Results of the S2 and L1 trials contribute to a body of research investigating the action of ANC in vision physiology (for a review, see Tremblay and Kalt29) and especially in relation to night vision and bilberry ANC (for a review, see Canter and Ernst2). Although S2 and L1 employed nonpurified blueberry products, the hypothesis underlying the design of the trials was that ANC was the blueberry component that could affect human vision because other abundant BJ components, including organic acids, simple sugars, and polysaccharides, have no demonstrated effect on vision. Dose and Treatment Regimen. Together the S2 and L1 studies spanned a wide range of exposure to ANCs. The S2 trial delivered in BJ 5.69 g C3g eq of ANC in 21 days, whereas the capsule delivered 199 mg C3g eq (or 3.4% of BJ dose) in the same period. In L1, BJ delivered a total of 29.1 g of ANCs in 84 days. The high daily dose of BJ in S2 and L1 (Table 1) is comparable to ANC doses used in other studies.30,31 The design of S2 and L1 did not test for acute effects because the time between product ingestion and vision testing was not controlled. Whether subjects were aware of the test product identity should be considered, because vision testing is psychophysical and can be susceptible to bias. In S2, subjects were informed that the BJ and the BJ powder capsule were the same product in two formulations and doses. Also in S2, the BJ powder in the capsule was deemed indistinguishable from the red beet powder capsule placebo. Many subjects in S2 and L1 had never previously consumed single-strength BJ and were not familiar with its sensory qualities. There was at least 8 weeks between when subjects consumed BJ or the placebo juice. Subjects had no opportunity to consume BJ and placebo juice at the same time.

a

DACRT, dark adaptation cone rod transition. bDAAUC, area under the curve, 9−30 min. cDAt90%, time to reach 90% of asymptote. dDAthr, final dark threshold, last 5 min. ecd/m2, candela per square meter.

the 4 or 8 week washout was significantly different from the placebo (Figure 3). PCA biplots were used to collectively illustrate possible relationships among the vision test results and dietary treatments. The S2 biplot accounted for 93% of the total variation (Figure 4A) with 63% in the first score (vertical axis) and 30% in the second score (horizontal axis). The L1 biplot accounted for 81% of the variation with 52% in the first score and 29% in the second score (Figure 4B). In both S2 and L1 the vector distances from each vision test (CS, DAthr, MSR, and VA) to the origin were quite similar, indicating that each of the four vision tests contributed roughly equally to the total variation illustrated by the biplots. In S2 the Euclidian distances between the dietary treatments distinguished the low-dose blueberry capsule (C) and the highdose BJ (B) from the placebo (P) along the MSR axis (Figure 4 A; solid line ellipse for blueberry products and washouts, dashed line ellipse for placebo treatment and washout). The capsule washout (Cw) and BJ washout (Bw) were also associated with low MSR values, suggesting a persistence of the effect during the washout. In the L1 biplot, the BJ and placebo treatments were well separated along the MSR axis (Figure 4A; solid line ellipse for BJ and BJ washout results, dashed line for placebo and placebo washout results). In both S2 and L1 the blueberry-related coordinates occurred in the E



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Figure 2. Macular stress recovery (MSR) test results defined as time (s) to recover visual acuity after retinal photobleaching in a short trial testing two blueberry products and placebo (S2) with n = 72. Mean MSR times ± SEM are shown for the pretreatment (no dietary products) and then after 3 weeks of dietary intervention and 3 weeks of washout. Means are back-transformed from log values which were used for ANOVA. These backtransformed values are plotted on a log scale with back transformed values shown.

Figure 3. Macular stress recovery (MSR) test results defined as time (s) to recover visual acuity after retinal photobleaching in a long trial testing one blueberry product and placebo (L1) with n = 59. Mean MSR times ± SEM are shown for the pretreatment (no dietary products) and then after 8 and 12 weeks of dietary intervention and 4 and 8 weeks of washout. Means are back-transformed from log values which were used for ANOVA. These back-transformed values are plotted on a log scale with back transformed values shown.

Vision Tests. DA. There was no evidence of improved DA with ANC intake in either S2 or L1. In S2 however, a significant treatment effect on DAthr (Table 3) supported some previous studies11,32−34 wherein ANC intake reduced the final dark threshold light intensity. Although not significant, the 3 week washout in S2 also supported this trend (Table 3). Whereas

this suggests that ANC improved the sensitivity of the darkadapted retina, the effect size (0.5 for treatment period; 0.25 for washout) was moderate. Also, there were no improvements in any other correlated measures of DA (DARCT, DAAUC, DAt90%). Furthermore, the L1 trial failed to show a similar trend for DAthr, despite a longer intervention with a high ANC dose. It is F



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Table 5. Visual Acuity and Contrast Sensitivity in the Dark after 30 min of Dark Adaptation in a Trial (S2) with a Short (3 Week) Intervention with a High Dose (Juice) and a Low Dose (Capsule) of Anthocyanins in Blueberry Products visual acuity (log minimal angle of resolution) juice capsule placebo grand mean SEM F prob, treatment effect size F prob, capsule vs juice F prob, placebo vs capsule and juice juice capsule placebo grand mean SEM F prob, washout effect size F prob, capsule vs juice F prob, placebo vs capsule and juice

Mean, 3 Week Treatment 0.41 0.41 0.41 0.41 0.005 ns 0.00

Mean, 3 Week Washout 0.39 0.40 0.41 0.40 0.004 0.014 0.58 0.022 0.042

Table 6. Visual Acuity and Contrast Sensitivity in the Dark after 30 min Dark Adaptation in a Trial (L1) with a Long Intervention Testing Blueberry Juice with a Juice Placebo visual acuity (log minimal angle of resolution)

contrast sensitivity (%) juice placebo grand mean SEM treatment effect size

3.03 3.03 3.17 3.08 0.060 ns 0.27

juice placebo grand mean SEM treatment effect size

2.89 2.76 2.82 2.82 0.060 ns 0.25


worth noting that this improvement in the DAthr in S2 was approximately 0.05 log10 cd/m2 (Tables 3 and 4) and is a difference that can be detected only under controlled conditions and would be imperceptible in real life. Variation in the DAthr means in S2 (SEM = 0.014) and L1 (SEM = 0.024) was of the same order of magnitude, suggesting that differences in the DA testing method were not a factor in the difference in DAthr values between the two trials (Tables 3 and 4). Together the S2 and L1 DAthr results do not support the early European literature that report improvement in DA parameters with ANCs,11,32−34 results which Canter and Ernst2 suggest may be influenced by weak experimental design. When Levy5 used double-blinded, crossover, randomized control protocols, they failed to show improvement in DA among young normal-sighted men in a study using different ANC doses and durations. Muth et al.7 also used rigorous protocols and found no effects among young normal-sighted men in other attributes of scotopic vision (VA and CS), but they did not test DA specifically. The S2 and L1 trials were designed to test for ANC effects on DA, CS, and VA in a population spanning 30 years in age (35−65, Table 1) across both genders. Subjects older than 65 years were not included in the study because the incidence of confounding vision health conditions such as subclinical cataracts and retinal dysfunctions is more common in this population group. CS and VA. The lack of an ANC effect on either CS or VA in both S2 and L1 may not be surprising because both of these tests rely more on the photoreceptor density and their underlying synaptic circuitry and less on mechanisms where ANC have been shown to be influential.35,36 A few studies have proposed an effect of ANCs on VA and CS,17,37 whereas randomized controlled design studies report negative results for ANC effects on CS6−8 and on VA.7 Notably, symptoms of


8 Week Treatment 0.47 0.45 0.46

contrast sensitivity (%) 3.03 3.36 3.20

0.011 ns 0.23 12 Week Treatment 0.45 0.44 0.44

0.174 ns 0.25

0.011 ns 0.00 4 Week Washout 0.44 0.43 0.43

0.143 ns 0.17

0.011 NS 0.12 8 Week Washout 0.43 0.43 0.43

0.112 NS 0.01

0.011 ns 0.00

2.78 2.97 2.88

2.66 2.65 2.65

2.73 2.51 2.62 0.139 ns 0.20

asthenopia (visual fatigue) that can relate to changes in VA and CS were reported to be reduced, on the basis of a subjective assessment.3 MSR. The MSR test, which measured the recovery time of visual acuity after controlled photoreceptor bleaching, depends heavily on the regeneration of rhodopsin38 in a way that is similar to DA. The degree of photoreceptor bleaching for the MSR test was greater than for DA testing in both S2 and L1. The MSR test measures the recovery of prebleaching acuity involving cone photoreceptors, whereas the DA test assesses the sensitivity of rod photoreceptors. Although photostress recovery time increases with subject age, it is independent of pupil size, ametropia (refractive error), and VA.38 The MSR test, which is also known as the retinal dazzling or blinding test, has been used clinically to discriminate retinal diseases from conditions of the optic nerve.39−41 In S2 and L1 ANC intake significantly reduced the MSR time; however, the effect size was moderate, ranging between 0.37 and 0.42. In S2 the improvement in MSR was observed in just 3 weeks, and this trend persisted during the 3 week washout. MSR improvement in S2 was found with both the high (271 mg C3g eq daily) and a low (7.11 mg C3g eq daily) ANC dose (Table 1; Figure 2). In L1, which used a high ANC dose (346 mg C3g daily), a similar reduction in MSR time was seen at 8 weeks (P = 0.027) and at 12 weeks (P = 0.03) and with a nonsignificant trend persisting at 4 weeks of washout. G



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(solid line ellipse) were clustered along the MSR axis in both S2 and L1. Furthermore, the quite random positioning of the blueberry treatment groups along the DAthr, VA, or CS vectors in both S2 and L1 suggests that the absence of statistical significance for these three vision tests is not due to an unpowered study but rather to the absence of correlation between these three vision tests and the intake of ANC. Study Design. Canter and Ernst2 cited the need for rigorous clinical research designs and controls to examine possible ANC effects on vision, which the S2 and L1 trials attempted to do. Study volunteers were middle-aged and had undergone detailed prescreening prior to enrollment to ensure that the study population was made up of normal-sighted individuals who were free of vision conditions or pathologies that could influence the vision testing. Rigor in the S2 and L1 study designs included double-blinding by vision testers (to the current usage) and subjects (to the identity) of test products or washouts. The use of a complete crossover and the use of a placebo also contributed to the strength of the study design. Conducting vision testing before the start of the dietary intervention (pretreatment) mitigated any effect of the subject learning the new vision tests, and in L1 subjects were already familiar with the tests due to their prior participation in S2. Covariate correction addressed possible effects unrelated to the dietary intervention that may have affected vision test performance. A challenge in investigating phytochemical interventions is that beneficial effects may not occur in healthy well-nourished populations who have access to a variety of plant-based foods, whereas effects may be still be demonstrable in a phytochemically deficient population. And within the well-nourished Western population, volunteers for nutritional studies are more likely to be nutrition-conscious and consuming a diet containing fruits and vegetables. Results of our study thus contribute to the evidence that nutritional studies related to vision should either target more vulnerable populations or utilize other investigational strategies. ANC intervention studies that target a population with an existing vision pathology may yield more positive outcomes; examples can already be found in relation to glaucoma symptoms44,45 and vascular pathologies.46,47 The notion that suboptimal vision may be improved by dietary ANCs has been proposed recently by Shin et al.48 We are currently investigating ANC intake effects in individuals with impaired vision. Also, large-scale epidemiological studies have associated ANC intake with risk reduction for cardiovascular factors49,50 and type 2 diabetes,51 and therefore such an approach is warranted to examine vision health and performance. Although S2 and L1 failed to demonstrate functional ANC benefits, we have recently reported a potent action of ANC in protecting rats from retinal injury created by exposure to intense light.19 In this study retinal damage, which was measured objectively by electroretinography and retinal tissue histology, was reduced from 60% in the placebo-fed rats to 20% in BJ-enriched diet. Animal studies benefit significantly from their control of genetics, lifestyle, diet, and the availability of many more research models. Furthermore, laboratory animals that have had limited or no previous exposure to ANCs may be predisposed to greater ANC action compared to humans, where ANC may be consumed regularly and persist in vivo for a long period.42,43 The S2 and L1 trials were rigorously designed to test for ANC effects on the performance of subjects in common

Figure 4. Principal component analyses for two clinical trials comparing results for measures of night vision. One trial used a short (3 weeks) intervention and tested two blueberry products (S2) (A); the other trial used a longer intervention (up to 12 weeks) and tested one blueberry product (L1) (B). Panel A and B abbreviations for vision tests: DAthr, dark threshold; CS, contrast sensitivity; MSR, macular stress recovery; VA, visual acuity. Panel A interventions: B, blueberry juice, 3 weeks; BW, blueberry juice, 3 week washout; C, blueberry capsule, 3 weeks; CW, blueberry capsule, 3 week washout; P, placebo, 3 weeks; PW, placebo, 3 week washout. Panel B interventions: B8, blueberry juice, 8 weeks; B12, blueberry juice, 12 weeks; Bw4, blueberry juice, 4 week washout; Bw8, blueberry juice, 8 week washout; P8, placebo juice, 8 weeks; P12, placebo juice, 12 weeks; Pw4, placebo juice, 4 week washout; Pw8, placebo juice, 8 week washout.

Together the results suggest that ongoing exposure to even a small ANC dose improved MSR time and that the effect persisted for a time after the cessation of ANC intake. This result may be related to the occurrence of ANC glycosides in tissues42 including eye tissues1,21 and the persistence of ANC metabolites in enteric circulation.43 A diverse array of phase II metabolites of ANC have recently been reported to persist in urine.43 It is not known whether phase II metabolites of ANC affect MSR. Multivariate PCA. The multivariate PCA explained the MSR test results with respect to the variation accounted for in S2 (Figure 4A) and L1 (Figure 4B). The MSR vector correlated poorly with the other three vision test vectors that together contributed to a large proportion of the total variation. Whereas all four vision tests (DAthr, MSR, VA, and CS) were poorly correlated, the coordinates of the blueberry treatment groups H



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(6) Mayser, H.; Wilhelm, H. Effects of anthocyanosides on contrast vision [abstract]. Invest. Ophth. Vis. Sci. 2001, 42, 348. (7) Muth, E.; Laurent, J.; Jasper, P. The effect of bilberry nutritional supplementation on night visual acuity and contrast sensitivity. Altern. Med. Rev. 2000, 5, 164−173. (8) Zadok, D.; Levy, Y.; Glovinsky, Y. The effect of anthocyanosides in a multiple oral dose on night vision. Eye 1999, 13 (Pt 6), 734−736. (9) Jayle, G.; Aubert, L. Action of anthocyanin glycosides on the scotopic and mesopic vision of the normal subject. Therapie 1964, 19, 171−185. (10) Alfieri, R.; Sole, P. Influence of anthocyanosides, in oralperilingual administration, on the adapto-electroretinogram (AERG) in red light in humans. C. R. Seances Soc. Biol. Fil. 1966, 160, 1590− 1593. (11) Belleoud, L.; Leluan, D.; Boyer, Y. Etude des effets des glucosides d’anthocyan sur la vision nocturne des controleurs d’aerodrome. Rev. Med. Aeronautique Spatiale 1966, 18, 3−7. (12) Jayle, G.; Aubry, M.; Gavini, H.; Braccini, G.; Baume, C. D. I. Study concerning the action of anthocyanoside extracts of Vaccinium myrtillus on night vision. Ann. Ocul. 1965, 198, 556−562. (13) Ponte, F.; Lauricella, M. Effect of Vaccinium myrtillus total extract on the recovery in the dark of the human electroretinogram. Atti VII Simposio; ISCERG: Istanbul, Turkey, 1969; pp 335−336. (14) Sala, D.; Rossi, P.; Rolando, S. Effetto degli antocianosidi sulle “performances” visive alle basse luminanze. Minerva Ophtalmol. 1979, 21, 283−285. (15) Sbrozzi, F.; Landini, J.; Zago, M. Night vision affected by anthocyanosides. An electoretinographic test. Minerva Ophtalmol. 1983, 24, 189−193. (16) Magnasco, A.; Zingirian, M. Influenza degli antocianosidi sulla soglia retinica differnziale mesopica [Influence of anthocyanosides on the mesopic differential threshold of the retina]. Ann. Ottamol. Clin. Ocul. 1966, 92, 188−193. (17) Lee, J.; Lee, H.; Kim, C.; Hong, Y.; Choe, C.; You, T.; Seong, G. Purified high-dose anthocyanoside oligomer administration improves nocturnal vision and clinical symptoms in myopia subjects. Br. J. Nutr. 2005, 93, 895−899. (18) Milbury, P.; Graf, B.; Curran-Celentano, J.; Blumberg, J. Bilberry (Vaccinium myrtillus) anthocyanins modulate heme oxygenase-1 and glutathione S-transferase-pi expression in ARPE-19 cells. Invest. Ophth. Vis. Sci. 2007, 48, 2343−2349. (19) Tremblay, F.; Waterhouse, J.; Nason, J.; Kalt, W. Prophylactic neuroprotection by blueberry-enriched diet in a rat model of lightinduced retinopathy. J. Nutr. Biochem. 2013, 24, 647−655. (20) Liu, Y.; Song, X.; Han, Y.; Zhou, F.; Zhang, D.; Ji, B.; Hu, J.; Lv, Y.; Cai, S.; Wei, Y.; Gao, F.; Jia, X. Identification of anthocyanin components of wild Chinese blueberries and amelioration of lightinduced retinal damage in pigmented rabbit using whole berries. J. Agric. Food Chem. 2010, 59, 356−363. (21) Matsumoto, H.; Nakamura, Y.; Iida, H.; Ito, K.; Ohguro, H. Comparative assessment of distribution of blackcurrant anthocyanins in rabbit and rat ocular tissues. Exp. Eye Res. 2006, 83, 348−356. (22) Clifford, M. N. Review: Anthocyanins − nature, occurrence and dietary burden. J. Sci. Food Agric. 2000, 80, 1063−1072. (23) Forney, C. F.; Kalt, W.; Jordan, M. A.; Vinqvist-Tymchuk, M. R.; Fillmore, S. A. E. Compositional changes in blueberry and cranberry fruit during ripening. Acta Hortic. (ISHS) 2012, 926, 331− 337. (24) Wrolstad, R.; Durst, R.; Lee, J. Tracking color and pigment changes in anthocyanin products. Trends Food Sci. Technol. 2005, 16, 423−428. (25) U.S. Department of Agriculture. National Nutrient Database for Standard Reference, release 25, 2012. (26) Ferris, F. L., 3rd; Freidlin, V.; Kassoff, A.; Green, S. B.; Milton, R.C. Relative letter and position difficulty on visual acuity charts from the Early Treatment Diabetic Retinopathy Study. Am. J. Ophthalmol. 1993, 116, 735−740. (27) Ginsburg, A. P. Contrast sensitivity and functional vision. Int. Ophthalmol. Clin. 2003, 43, 5−15.

psychophysical tests that measure visual function in the dark. ANC intervention had, at best, a moderate effect on the rate of recovery after retinal photobleaching. The magnitude of the effect would not likely translate into subjectively detectable improvement in everyday visual performance. There was no effect of ANC intake on dark adaptation and scotopic vision. At this juncture, with the limited positive results from welldesigned human interventional studies in normal populations, it may be useful to investigate the role of ANC in vision using an epidemiological approach or in interventional studies that target specific pathologies or experimental models.

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AUTHOR INFORMATION

Corresponding Author

*(W.K.) Phone: (902) 365-8561. Fax: (902) 365-8455. E-mail: [email protected]. Author Contributions

W.Kalt, J.E.M., and F.T. contributed to the project concept and study design, development of the overall research plan, and oversight in data analysis. F.T. developed the psychophysical tests. J.E.M. was study coordinator with activities in hands-on data collection, coordination with vision testers, and data analysis. S.A.E.F. was responsible for the statistical design and analysis. W.K. wrote the paper. W.K. and F.T. had primary responsibility for the final content. All authors read and approved the final manuscript. Funding

This research was funded by Agriculture and Agri-Food Canada and the U.S. Highbush Blueberry Council. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Vision testers: S2, Jane Carver, Peter Wightman; L1, Bradley Monro. Clinical screening for enrollment: Joan Parkinson, Darren Behn. Technical support: Melinda Vinqvist-Tymchuk.

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ABBREVIATIONS USED ANC, anthocyanin; BJ, blueberry juice; cd/m2, candela per meter squared; CS, contrast sensitivity; C3g eq, cyanidin 3glucoside equivalents; DA, dark adaptometry; DAAUC, area under the curve 9−30 min; DACRT, cone rod transition; DAt90%, time to reach 90% of asymptote; DAthr, final threshold, last 5 min; L1, long trial, one blueberry product; MSR, macular stress recovery; PCA, principle component analysis; S2, short trial, two blueberry products; VA, visual acuity

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REFERENCES

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