Acta Ophthalmologica 2014
The short-term effect of flavonoid-rich dark chocolate on retinal vessel diameter in glaucoma patients and age-matched controls Naim Terai, Alexandra Gedenk, Eberhard Spoerl, Lutz E Pillunat and Richard Stodtmeister Department of Ophthalmology, University Hospital Dresden, Dresden, Germany
ABSTRACT. Purpose: To investigate the effect of flavonoid-rich dark chocolate and nonflavonoid-rich white chocolate on retinal vessel diameter in glaucoma patients and age-matched controls. Methods: Thirty glaucoma patients and 30 age-matched subjects were assigned to dark or white chocolate by randomization with forced equal distribution. The number in each of the four groups was 15. Measured parameters included systemic blood pressure (BP), blood glucose levels, static retinal vessel analysis, as measured by central retinal artery equivalent (CRAE) (which relates to the diameter of the central retinal artery), central retinal vein equivalent (CRVE) (which relates to the diameter of central retinal vein) and the arterio-venous ratio (AVR), which represents the CRAE/CRVE ratio, dynamic retinal vessel analysis as measured by the change in vessel diameter in response to flicker light stimulation. Three recording cycles from each were averaged. Results: Blood pressure parameters (systolic BP, diastolic BP and pulse), IOP and blood glucose levels did not differ significantly between both groups before and after consumption of white or dark chocolate. Static vessel analysis did not show any significant changes in CRAE, CRVE or AVR before and after dark or white chocolate in both groups (p > 0.05).Mean dilatation of the venules in the control group was 3.2 0.9 % before dark chocolate and 4.2 1.4 % after dark chocolate intake, which was statistically significantly different (p = 0.01). Mean dilatation of the arterioles in the control group was 2.8 1.8 % before dark chocolate and 3.5 1.8 % after dark chocolate intake with a trend to statistical significance (p = 0.14), but not reaching the significance level. Mean diameter changes in the glaucoma group did not show any significant differences after dark chocolate consumption. Conclusion: The present study showed a significant improvement of venous vasodilatation 2 hr after dark chocolate intake in the control group, but not in the glaucoma group. This effect might be indicative of an increased bioavailability of nitric oxide (NO) after dark chocolate consumption. The lack of finding a significant venous response after dark chocolate in the glaucoma group might be related to the already impaired endothelial function in these patients. Key words: dark chocolate – dynamic vessel analysis – flavonoids – glaucoma – nitric oxide
Acta Ophthalmol. ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12373
Introduction Recent studies have provided evidence that the consumption of flavonoid-rich dark chocolate induces beneficial effects on the vascular endothelial function, most probably by decreasing oxidative stress and increasing nitric oxide (NO) bioavailability leading to an improvement of endothelium-dependent vasorelaxation (Karim et al. 2000; Herrmann et al. 2006). Dietary inclusion of polyphenol-rich dark chocolate was suggested to efficiently reduce blood pressure and improve formation of vasodilatative NO in healthy subjects but also in patients with systemic endothelial dysfunction as seen in patients with congestive heart failure (Flammer et al. 2012). Glaucoma is characterized by a slowly progressive remodelling of the optic nerve head (ONH) and a loss of retinal fibre layer in combination with visual field changes corresponding to increased excavation of the optic disc (Quigley et al. 1993). It is well known that an elevated IOP is the most important risk factor for the disease. However, other factors than elevated IOP may contribute to glaucomatous damage. One mechanism being postulated to be causative for primary open-angle glaucoma (POAG) is vascular dysfunction leading to ischaemia of the ONH. An imbalance between endothelin-1 (ET-1) and nitric oxide (NO) that is characteristic of endothelial dysfunction has been reported in glaucoma. Several other studies also indicated a comprised availability of NO in glaucoma (Resch et al. 2009). Although it is known that flavonoids augment endothelial nitric oxide
1
Acta Ophthalmologica 2014
synthase (NOS) and thereby NO to improve endothelium-dependent vasorelaxation, to the best of (our) knowledge, there are no data about in vivo effects on ocular blood flow even though it is widely assumed that flavonoids might have beneficial therapeutical effects on diseases like glaucoma via the abovementioned NO pathway and their antioxidative effect (Flammer et al. 2013). Against this background, it was the aim of the present study to investigate whether (i) there is a difference in vascular response after consumption of flavonoidcontaining dark chocolate or non-flavonoid-containing white chocolate and whether (ii) there is a difference in the vascular response in glaucoma patients compared to age-matched subjects. To do so, we employed the Dynamic VesselAnalyzer (Imedos,Jena,Germany).
Methods Subjects
The study protocol was approved by the Ethics Committee of University Hospital Dresden and adheres to the tenets of the Declaration of Helsinki. All subjects signed informed consent before participating in this study. This study was conducted as a randomized, single-blinded prospective placebo-controlled study. Included were glaucoma patients and controls of >50 years of age. Glaucoma was defined from optic nerve head damage and visual field damage typical in glaucoma patients. Exclusion criteria in glaucoma patients and controls were as follows: diabetes, age-related macular degeneration (ARMD) and other ocular diseases, previous intraocular surgeries and/or retinal laser photocoagulation, ocular injuries, or lens/corneal opacities and smokers. Patients with migraine, epilepsy, uncontrolled arterial hypertension and prior (0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05
20 (66.7%) 2 (6.7%) 0 0 0 0 6 (20%) 3 (10%)
20 (66.7%) 7 (23.7%) 1 (3.3%) 5 (16.7%) 0 1 (3.3%) 8 (26.7%) 0
1.0 0.071 0.313 0.020 1.0 0.313 0.076 0.076
10 (33.3%) 4 (13.3%) 8 (26.7%) 7 (23.3%) 4 (13.3%) 4 (10%)
10 (33.3%) 10 (33.3%) 7 (23.3%) 8 (26.7%) 10 (33.3%) 7 (23.3%)
1.0 0.067 0.766 0.766 0.067 0.166
Bold value indicates statistical significance.
glaucoma group were 2.27 0.39 mm² (optic disc size), 1.11 0.39 mm² (rim size area) and 0.25 0.14 mm³ (rim size volume). All glaucoma patients were under topical antiglaucoma medication. Both subgroups of glaucoma patients were comparable according the distribution, type and number of antiglaucoma medications. Blood pressure parameters, IOP and blood glucose levels did not show statistically significant differences after dark or white chocolate consumption in either of the two groups (all p > 0.05), Table 1. Tables 2 and 3 show the changes of static (CRAE, CRVE, AV ratio) and dynamic (dilatation and constriction of arterioles in percentage, dilatation of venules in percentage) parameters before and after white and dark chocolate intake in the control and glaucoma group. Mean dilatation of the venules was 3.2 0.9 % before dark chocolate and 4.2 1.4 % after dark chocolate intake, which was statistically significantly different (p = 0.01). Table 4 shows the difference of static and dynamic parameters as an increase after intake for the controls and the glaucoma group. Figure 1 illustrates the dilatation of the venules in percentage before and after dark chocolate intake in the control and glaucoma group.
Discussion The present study showed a significant improvement of venous vasodilatation 2 hr after flavonoid-rich dark chocolate intake in the control group, but not in the glaucoma group. Although showing a trend to statistical significance, the arterioles did not show any significant change in diameter in the dynamic vessel analysis. The minimal flicker-induced dilatation of thearteriolesobservedismostlikelydueto the fact that our study patients were of advanced age, wherein vessels have become too sclerotic to undergo changes in diameter. Age-related effects on the vasculature consist of a remodelling of the vascular wall, leading to atherosclerosis and to a stiffening of the arterioles (Toda 2012), while age-related changes in the vascular response are, perhaps, due to endothelial dysfunction. The capacity of the vascular endothelium to generate nitric oxide (NO) also declines with advancing age so that especially the arterial vascular reactivity decreases. It is well known that flicker responses are diminished in glaucoma patients (Garh€ ofer et al. 2004; Gugleta et al. 2012). However, looking at the flicker-induced vasodilatation responses in our glaucoma patients showed that they were not reduced. Garh€ ofer and coworkers observed flicker responses of 0.2–1.1% in the venules and 0.3–1.8% in
3
Acta Ophthalmologica 2014
Table 2. Static and dynamic vessel parameters before and after white or dark chocolate intake in the control group, Values are means s (= standard deviation). White chocolate Control subjects n
Before intake 15 Mean s
CRAE (lm) CRVE (lm) AV Ratio Dilat. Arterioles (%) Constr. Arterioles (%) Dilat. Venules (%)
183 208 0.88 2.3 0.2 3.3
Dark chocolate After intake 15 Mean s
12 20 0.07 1.5 0.8 2.0
183 208 0.88 2.6 0.3 3.4
12 20 0.07 1.9 1.2 1.9
p
Before intake 15 Mean s
0.94 0.80 0.76 0.49 0.76 0.65
18 0 208 0.87 2.8 0.1 3.2
16 21 0.06 1.8 2.2 0.9
After intake 15 Mean s 179 205 0.87 3.5 0.6 4.2
13 17 0.06 1.8 1.1 1.4
p 0.37 0.17 0.43 0.14 0.14 0.01
Table 3. Static and dynamic vessel parameters before and after white or dark chocolate intake in the glaucoma group, Values are means s (=standard deviation). White chocolate Glaucoma patients n
Before intake 15 Mean s
CRAE (lm) CRVE (lm) AV Ratio Dilat. Arterioles (%) Constr. Arterioles (%) Dilat. Venules (%)
173 206 0.84 3.6 0.1 4.8
Dark chocolate After intake 15 Mean s
19 17 0.07 2.4 1.2 2.3
171 206 0.83 3.0 -0.8 4.2
18 15 0.06 1.7 1.3 1.8
p
Before intake 15 Mean s
0.25 0.75 0.26 0.11 0.08 0.11
170 195 0.87 3.2 0.4 4.5
Table 4. Differences of static and dynamic parameters as an increase after dark or white chocolate in control and glaucoma group, Values are means s (= standard deviation). Differences of parameters Parameter after intake – Parameter before intake Positive value: Increase after intake Controls Mean s CRAE white chocolate (lm) CRAE dark chocolate (lm) CRVE white chocolate (lm) CRVE dark chocalate (lm) AVR white chocolate AVR dark chocolate Dilation of arterioles white ch.(%) Dilation of arterioles dark ch. (%) Constriction of arterioles white ch. (%) Constriction of arterioles dark ch. (%) Dilation of venules white ch. (%) Dilation of venules dark ch.(%)
the arterioles in early glaucoma patients (Garh€ ofer et al. 2004). Similar results were obtained by Gugleta and colleagues (Gugleta et al. 2012). Despite small differences in methodological issues, the two studies allow a good comparability to our study. However, the patients in the above-mentioned studies were not under topical antiglaucoma medication but instead had a washout period for 2– 4 weeks before being included in the
4
0 1 0 2 0.00 0.00 0.3 0.7 0.1 0.7 0.1 1.0
5 6 4 6 0.2 0.02 1.5 1.8 1.3 1.8 1.2 1.3
Glaucoma Mean s
p
1 2 0 2 0.00 0.00 0.7 0.2 0.9 0.0 0.6 0.5
0.38 0.91 0.94 1.00 0.31 0.93 0.10 0.39 0.20 0.27 0.13 0.34
5 5 4 5 0.2 0.2 1.5 1.5 1.8 1.6 1.3 1.6
study. We therefore assume a pharmacological effect which might have been responsible for not finding a diminished response in our glaucoma patients. Further studies are certainly needed to investigate this topic in more detail. The improvement of the venous dilatation which we observed can most probably be attributed to the action of flavonols which – among other effects – also augment endothelial NOS 3 and
11 17 0.06 2.3 1.5 2.2
After intake 15 Mean s 168 192 0.88 3.3 0.4 5.0
9 15 0.07 1.9 1.3 2.1
p 0.25 0.10 0.65 0.64 0.95 0.25
thereby NO leading to an improvement of endothelium-dependent vasorelaxation (Duffy & Vita 2003). To the best of our knowledge, our in vivo study is the first to investigate the effect of flavonols on ocular blood flow in glaucoma patients in a randomized, placebo-controlled study design. Therefore, unfortunately, there are no comparable study data in the literature. However, the vasodilatative effect of flavonoids is well known from cardiological studies. Taubert and colleagues using a randomized, controlled, investigator-blinded study showed a reduction of blood pressure and an improved formation of vasodilatative NO (S-nitrosoglutathione) in the plasma in patients with untreated upperrange prehypertension and hypertension stage 1 after intake of 30 mg of polyphenol-containing dark chocolate for 18 weeks (Taubert et al. 2007). A study of Flammer and collaborators detected an improvement of vascular function in the short term (2 h after ingestion) but also in the long term (4 weeks) after regular flavonol-rich dark chocolate intake in patients with congestive heart failure using flow-mediated vasodilatation of the brachial artery. This effect was accompanied by an inhibition of platelet function in these patients (Flammer et al. 2013). However, because of
Acta Ophthalmologica 2014
Fig. 1. Dilation of retinal vessels to flicker stimulation before and after intake of dark chocolate.
differences in methodology and vascular beds, comparisons of these latter two studies to ours are difficult and limited. Another recent study of Flammer and co-workers investigating the short-term effect (after 2 hr) of 40 g of flavonoidrich dark chocolate on coronary vascular and platelet function in 22 heart transplant recipient patients following heart transplantation showed a significant increase in coronary artery diameter (Flammer et al. 2007). In their study age of the patients was 51 years in the dark chocolate and 55 years in the control group and therefore much younger than in our study groups (64 and 65 years). It may therefore be hypothesized that remodelling processes were less advanced than in our patients because we did not observe any significant change of arterial diameter after dark chocolate intake. The present study has a limitation. DVA only provides information regarding vessel diameter and retinal vessel reactivity to flicker stimulation. For determination of changes in blood flow per se, information regarding blood velocity would be required. In principle, this information could be gained using laser Doppler flowmetry (LDF) or laser Doppler velocimetry (LDV). Bi-directional optical laser Doppler tomography could be used to address this issue in future studies, although the reproducibility of that method still needs further investigated (Wang et al. 2007; Pemp et al. 2013). Taking together, the present study indicated an improvement of endothelial
function 2 hr after flavonoid-dark chocolate intake leading to an increase in vasodilatation of the venules. This effect seems to be elicited by augmentation of NO production and NO bioavailability at the endothelium of retinal vessels by the action of flavonols. Further studies with longer follow-up periods are certainly needed to investigate whether our preliminary results are confirmed and to proof whether flavonols might have a beneficial therapeutical potency for the treatment of glaucoma.
References Duffy SJ & Vita JA (2003): Effects of phenolics on endothelial function. Curr Opin Lipidol 14: 21– 27. Flammer AJ, Hermann F, Sudano I et al. (2007): Dark chocolate improves coronary vasomotion and reduces platelet reactivity. Circulation 116: 2376–2382 Flammer AJ, Sudano I, Wolfrum M et al. (2012): Cardiovascular effects of flavonol-rich chocolate in patients with heart failure. Euro Heart Journal 33: 2172–2180. Flammer J, Mozaffarieh M & Bebie H (2013): Basic sciences in ophthalmology. Heidelberg, Germany: Springer Verlag 164–165. Garhofer G, Bek T, Boehm AG et al. (2010): Use of the retinal vessel analyzer in ocular blood flow research. Acta Ophthalmol 88: 717– 722. Garh€ ofer G, Zawinka C, Resch H et al. (2004): Response of retinal vessel diameters to flicker stimulation in patients with early open angle glaucoma. J Glaucoma 13: 340–344. Gugleta K, Kochkorov A, Waldmann N et al. (2012): Dynamics of retinal vessel response to flicker light in glaucoma patients and ocular hypertensives. Graefes Arch Clin Exp Ophthalmol 250: 589–594.
Herrmann F, Spieker LE, Ruschitzka F et al. (2006): Dark chocolate improves endothelial and platelet function. Heart 92: 119–120. Hubbard LD, Brothers RG, King WN et al. (1999): Methods for evaluation of retinal microvascular abnormalities associated with hypertension/sclerosis in the atherosclerosis risk in communities study. Ophthalmology 106: 2269–2280. Karim M, Mc Cormick K & Kappagoda CT (2000): Effects of cocao extracts on endothelium-dependent relaxation. J Nutr 130: 2105S– 2108S. Kur J, Newman EA & Chan-Ling T (2012): Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease. Prog Retin Eye Res 31: 377–406. Nagel E & Vilser W (2004): Flicker observation light induces diameter response in retinal arterioles: a clinical methodological study. Br J Ophthalmol 88: 54–56. Newman EA (2013): Functional hyperemia and mechanisms of neurovascular coupling in the retinal vasculature. J Cereb Blood Flow Metab 33: 1685–1695. Pemp B, Cherecheanu AP, Garhofer G et al. (2013): Calculation of central retinal artery diameters from non-invasive ocular haemodynamic measurements in type 1 diabetes patients. Acta Ophthalmol 91: e348–e352. Polak K, Dorner G, Kiss B, Kiss B, Polska E, Findl O, Rainer G, Eichler HG, Schmetterer L. (2000): Evaluation of the Zeiss retinal vessel analyser. Br J Ophthalmol 84: 1285–1290. Popa Cherecheanu A, Garhofer G, Schmidl D et al. (2013): Ocular perfusion pressure and ocular blood flow in glaucoma. Curr Opin Pharmacol 13: 36–42. Quigley HA, West SK, Munoz B et al. (1993): Examination methods for glaucoma prevalence surveys. Arch Ophthalmol 111: 1409–1415. Resch H, Garhofer G, Fuchsj€ ager-Mayrl G, Hommer A, Schmetterer L. (2009): Endothelial function in glaucoma. Acta Ophthalmol 87: 4– 12 Taubert D, Roesen R, Lehmann C et al. (2007): Effects of low habitual cacao intake on blood pressure and bioactive nitric oxide: a randomized controlled trial. JAMA 298: 49–60. Toda N (2012): Age-related changes in endothelial function and blood flow regulation. Pharmacol Ther 133: 159–176. Wang Y, Bower BA, Izatt JA et al. (2007): In vivo total retinal blood flow measurement by Fourier-domain optical coherence tomography. J Biomed Opt 12: 041215.
Received on October 2nd, 2013. Accepted on January 29th, 2014. Correspondence: Naim Terai, MD Department of Ophthalmology Carl Gustav Carus University Hospital Fetscherstr. 74 01307 Dresden, Germany Tel: +49 351 458 5091 Fax: +49 351 458 4335 Email: [email protected]
5
The short-term effect of flavonoid-rich dark chocolate on retinal vessel diameter in glaucoma patients and age-matched controls Naim Terai, Alexandra Gedenk, Eberhard Spoerl, Lutz E Pillunat and Richard Stodtmeister Department of Ophthalmology, University Hospital Dresden, Dresden, Germany
ABSTRACT. Purpose: To investigate the effect of flavonoid-rich dark chocolate and nonflavonoid-rich white chocolate on retinal vessel diameter in glaucoma patients and age-matched controls. Methods: Thirty glaucoma patients and 30 age-matched subjects were assigned to dark or white chocolate by randomization with forced equal distribution. The number in each of the four groups was 15. Measured parameters included systemic blood pressure (BP), blood glucose levels, static retinal vessel analysis, as measured by central retinal artery equivalent (CRAE) (which relates to the diameter of the central retinal artery), central retinal vein equivalent (CRVE) (which relates to the diameter of central retinal vein) and the arterio-venous ratio (AVR), which represents the CRAE/CRVE ratio, dynamic retinal vessel analysis as measured by the change in vessel diameter in response to flicker light stimulation. Three recording cycles from each were averaged. Results: Blood pressure parameters (systolic BP, diastolic BP and pulse), IOP and blood glucose levels did not differ significantly between both groups before and after consumption of white or dark chocolate. Static vessel analysis did not show any significant changes in CRAE, CRVE or AVR before and after dark or white chocolate in both groups (p > 0.05).Mean dilatation of the venules in the control group was 3.2 0.9 % before dark chocolate and 4.2 1.4 % after dark chocolate intake, which was statistically significantly different (p = 0.01). Mean dilatation of the arterioles in the control group was 2.8 1.8 % before dark chocolate and 3.5 1.8 % after dark chocolate intake with a trend to statistical significance (p = 0.14), but not reaching the significance level. Mean diameter changes in the glaucoma group did not show any significant differences after dark chocolate consumption. Conclusion: The present study showed a significant improvement of venous vasodilatation 2 hr after dark chocolate intake in the control group, but not in the glaucoma group. This effect might be indicative of an increased bioavailability of nitric oxide (NO) after dark chocolate consumption. The lack of finding a significant venous response after dark chocolate in the glaucoma group might be related to the already impaired endothelial function in these patients. Key words: dark chocolate – dynamic vessel analysis – flavonoids – glaucoma – nitric oxide
Acta Ophthalmol. ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12373
Introduction Recent studies have provided evidence that the consumption of flavonoid-rich dark chocolate induces beneficial effects on the vascular endothelial function, most probably by decreasing oxidative stress and increasing nitric oxide (NO) bioavailability leading to an improvement of endothelium-dependent vasorelaxation (Karim et al. 2000; Herrmann et al. 2006). Dietary inclusion of polyphenol-rich dark chocolate was suggested to efficiently reduce blood pressure and improve formation of vasodilatative NO in healthy subjects but also in patients with systemic endothelial dysfunction as seen in patients with congestive heart failure (Flammer et al. 2012). Glaucoma is characterized by a slowly progressive remodelling of the optic nerve head (ONH) and a loss of retinal fibre layer in combination with visual field changes corresponding to increased excavation of the optic disc (Quigley et al. 1993). It is well known that an elevated IOP is the most important risk factor for the disease. However, other factors than elevated IOP may contribute to glaucomatous damage. One mechanism being postulated to be causative for primary open-angle glaucoma (POAG) is vascular dysfunction leading to ischaemia of the ONH. An imbalance between endothelin-1 (ET-1) and nitric oxide (NO) that is characteristic of endothelial dysfunction has been reported in glaucoma. Several other studies also indicated a comprised availability of NO in glaucoma (Resch et al. 2009). Although it is known that flavonoids augment endothelial nitric oxide
1
Acta Ophthalmologica 2014
synthase (NOS) and thereby NO to improve endothelium-dependent vasorelaxation, to the best of (our) knowledge, there are no data about in vivo effects on ocular blood flow even though it is widely assumed that flavonoids might have beneficial therapeutical effects on diseases like glaucoma via the abovementioned NO pathway and their antioxidative effect (Flammer et al. 2013). Against this background, it was the aim of the present study to investigate whether (i) there is a difference in vascular response after consumption of flavonoidcontaining dark chocolate or non-flavonoid-containing white chocolate and whether (ii) there is a difference in the vascular response in glaucoma patients compared to age-matched subjects. To do so, we employed the Dynamic VesselAnalyzer (Imedos,Jena,Germany).
Methods Subjects
The study protocol was approved by the Ethics Committee of University Hospital Dresden and adheres to the tenets of the Declaration of Helsinki. All subjects signed informed consent before participating in this study. This study was conducted as a randomized, single-blinded prospective placebo-controlled study. Included were glaucoma patients and controls of >50 years of age. Glaucoma was defined from optic nerve head damage and visual field damage typical in glaucoma patients. Exclusion criteria in glaucoma patients and controls were as follows: diabetes, age-related macular degeneration (ARMD) and other ocular diseases, previous intraocular surgeries and/or retinal laser photocoagulation, ocular injuries, or lens/corneal opacities and smokers. Patients with migraine, epilepsy, uncontrolled arterial hypertension and prior (0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05
20 (66.7%) 2 (6.7%) 0 0 0 0 6 (20%) 3 (10%)
20 (66.7%) 7 (23.7%) 1 (3.3%) 5 (16.7%) 0 1 (3.3%) 8 (26.7%) 0
1.0 0.071 0.313 0.020 1.0 0.313 0.076 0.076
10 (33.3%) 4 (13.3%) 8 (26.7%) 7 (23.3%) 4 (13.3%) 4 (10%)
10 (33.3%) 10 (33.3%) 7 (23.3%) 8 (26.7%) 10 (33.3%) 7 (23.3%)
1.0 0.067 0.766 0.766 0.067 0.166
Bold value indicates statistical significance.
glaucoma group were 2.27 0.39 mm² (optic disc size), 1.11 0.39 mm² (rim size area) and 0.25 0.14 mm³ (rim size volume). All glaucoma patients were under topical antiglaucoma medication. Both subgroups of glaucoma patients were comparable according the distribution, type and number of antiglaucoma medications. Blood pressure parameters, IOP and blood glucose levels did not show statistically significant differences after dark or white chocolate consumption in either of the two groups (all p > 0.05), Table 1. Tables 2 and 3 show the changes of static (CRAE, CRVE, AV ratio) and dynamic (dilatation and constriction of arterioles in percentage, dilatation of venules in percentage) parameters before and after white and dark chocolate intake in the control and glaucoma group. Mean dilatation of the venules was 3.2 0.9 % before dark chocolate and 4.2 1.4 % after dark chocolate intake, which was statistically significantly different (p = 0.01). Table 4 shows the difference of static and dynamic parameters as an increase after intake for the controls and the glaucoma group. Figure 1 illustrates the dilatation of the venules in percentage before and after dark chocolate intake in the control and glaucoma group.
Discussion The present study showed a significant improvement of venous vasodilatation 2 hr after flavonoid-rich dark chocolate intake in the control group, but not in the glaucoma group. Although showing a trend to statistical significance, the arterioles did not show any significant change in diameter in the dynamic vessel analysis. The minimal flicker-induced dilatation of thearteriolesobservedismostlikelydueto the fact that our study patients were of advanced age, wherein vessels have become too sclerotic to undergo changes in diameter. Age-related effects on the vasculature consist of a remodelling of the vascular wall, leading to atherosclerosis and to a stiffening of the arterioles (Toda 2012), while age-related changes in the vascular response are, perhaps, due to endothelial dysfunction. The capacity of the vascular endothelium to generate nitric oxide (NO) also declines with advancing age so that especially the arterial vascular reactivity decreases. It is well known that flicker responses are diminished in glaucoma patients (Garh€ ofer et al. 2004; Gugleta et al. 2012). However, looking at the flicker-induced vasodilatation responses in our glaucoma patients showed that they were not reduced. Garh€ ofer and coworkers observed flicker responses of 0.2–1.1% in the venules and 0.3–1.8% in
3
Acta Ophthalmologica 2014
Table 2. Static and dynamic vessel parameters before and after white or dark chocolate intake in the control group, Values are means s (= standard deviation). White chocolate Control subjects n
Before intake 15 Mean s
CRAE (lm) CRVE (lm) AV Ratio Dilat. Arterioles (%) Constr. Arterioles (%) Dilat. Venules (%)
183 208 0.88 2.3 0.2 3.3
Dark chocolate After intake 15 Mean s
12 20 0.07 1.5 0.8 2.0
183 208 0.88 2.6 0.3 3.4
12 20 0.07 1.9 1.2 1.9
p
Before intake 15 Mean s
0.94 0.80 0.76 0.49 0.76 0.65
18 0 208 0.87 2.8 0.1 3.2
16 21 0.06 1.8 2.2 0.9
After intake 15 Mean s 179 205 0.87 3.5 0.6 4.2
13 17 0.06 1.8 1.1 1.4
p 0.37 0.17 0.43 0.14 0.14 0.01
Table 3. Static and dynamic vessel parameters before and after white or dark chocolate intake in the glaucoma group, Values are means s (=standard deviation). White chocolate Glaucoma patients n
Before intake 15 Mean s
CRAE (lm) CRVE (lm) AV Ratio Dilat. Arterioles (%) Constr. Arterioles (%) Dilat. Venules (%)
173 206 0.84 3.6 0.1 4.8
Dark chocolate After intake 15 Mean s
19 17 0.07 2.4 1.2 2.3
171 206 0.83 3.0 -0.8 4.2
18 15 0.06 1.7 1.3 1.8
p
Before intake 15 Mean s
0.25 0.75 0.26 0.11 0.08 0.11
170 195 0.87 3.2 0.4 4.5
Table 4. Differences of static and dynamic parameters as an increase after dark or white chocolate in control and glaucoma group, Values are means s (= standard deviation). Differences of parameters Parameter after intake – Parameter before intake Positive value: Increase after intake Controls Mean s CRAE white chocolate (lm) CRAE dark chocolate (lm) CRVE white chocolate (lm) CRVE dark chocalate (lm) AVR white chocolate AVR dark chocolate Dilation of arterioles white ch.(%) Dilation of arterioles dark ch. (%) Constriction of arterioles white ch. (%) Constriction of arterioles dark ch. (%) Dilation of venules white ch. (%) Dilation of venules dark ch.(%)
the arterioles in early glaucoma patients (Garh€ ofer et al. 2004). Similar results were obtained by Gugleta and colleagues (Gugleta et al. 2012). Despite small differences in methodological issues, the two studies allow a good comparability to our study. However, the patients in the above-mentioned studies were not under topical antiglaucoma medication but instead had a washout period for 2– 4 weeks before being included in the
4
0 1 0 2 0.00 0.00 0.3 0.7 0.1 0.7 0.1 1.0
5 6 4 6 0.2 0.02 1.5 1.8 1.3 1.8 1.2 1.3
Glaucoma Mean s
p
1 2 0 2 0.00 0.00 0.7 0.2 0.9 0.0 0.6 0.5
0.38 0.91 0.94 1.00 0.31 0.93 0.10 0.39 0.20 0.27 0.13 0.34
5 5 4 5 0.2 0.2 1.5 1.5 1.8 1.6 1.3 1.6
study. We therefore assume a pharmacological effect which might have been responsible for not finding a diminished response in our glaucoma patients. Further studies are certainly needed to investigate this topic in more detail. The improvement of the venous dilatation which we observed can most probably be attributed to the action of flavonols which – among other effects – also augment endothelial NOS 3 and
11 17 0.06 2.3 1.5 2.2
After intake 15 Mean s 168 192 0.88 3.3 0.4 5.0
9 15 0.07 1.9 1.3 2.1
p 0.25 0.10 0.65 0.64 0.95 0.25
thereby NO leading to an improvement of endothelium-dependent vasorelaxation (Duffy & Vita 2003). To the best of our knowledge, our in vivo study is the first to investigate the effect of flavonols on ocular blood flow in glaucoma patients in a randomized, placebo-controlled study design. Therefore, unfortunately, there are no comparable study data in the literature. However, the vasodilatative effect of flavonoids is well known from cardiological studies. Taubert and colleagues using a randomized, controlled, investigator-blinded study showed a reduction of blood pressure and an improved formation of vasodilatative NO (S-nitrosoglutathione) in the plasma in patients with untreated upperrange prehypertension and hypertension stage 1 after intake of 30 mg of polyphenol-containing dark chocolate for 18 weeks (Taubert et al. 2007). A study of Flammer and collaborators detected an improvement of vascular function in the short term (2 h after ingestion) but also in the long term (4 weeks) after regular flavonol-rich dark chocolate intake in patients with congestive heart failure using flow-mediated vasodilatation of the brachial artery. This effect was accompanied by an inhibition of platelet function in these patients (Flammer et al. 2013). However, because of
Acta Ophthalmologica 2014
Fig. 1. Dilation of retinal vessels to flicker stimulation before and after intake of dark chocolate.
differences in methodology and vascular beds, comparisons of these latter two studies to ours are difficult and limited. Another recent study of Flammer and co-workers investigating the short-term effect (after 2 hr) of 40 g of flavonoidrich dark chocolate on coronary vascular and platelet function in 22 heart transplant recipient patients following heart transplantation showed a significant increase in coronary artery diameter (Flammer et al. 2007). In their study age of the patients was 51 years in the dark chocolate and 55 years in the control group and therefore much younger than in our study groups (64 and 65 years). It may therefore be hypothesized that remodelling processes were less advanced than in our patients because we did not observe any significant change of arterial diameter after dark chocolate intake. The present study has a limitation. DVA only provides information regarding vessel diameter and retinal vessel reactivity to flicker stimulation. For determination of changes in blood flow per se, information regarding blood velocity would be required. In principle, this information could be gained using laser Doppler flowmetry (LDF) or laser Doppler velocimetry (LDV). Bi-directional optical laser Doppler tomography could be used to address this issue in future studies, although the reproducibility of that method still needs further investigated (Wang et al. 2007; Pemp et al. 2013). Taking together, the present study indicated an improvement of endothelial
function 2 hr after flavonoid-dark chocolate intake leading to an increase in vasodilatation of the venules. This effect seems to be elicited by augmentation of NO production and NO bioavailability at the endothelium of retinal vessels by the action of flavonols. Further studies with longer follow-up periods are certainly needed to investigate whether our preliminary results are confirmed and to proof whether flavonols might have a beneficial therapeutical potency for the treatment of glaucoma.
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Received on October 2nd, 2013. Accepted on January 29th, 2014. Correspondence: Naim Terai, MD Department of Ophthalmology Carl Gustav Carus University Hospital Fetscherstr. 74 01307 Dresden, Germany Tel: +49 351 458 5091 Fax: +49 351 458 4335 Email: [email protected]
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