Optometry (2009) 80, 695-701

Molecular medicine in ophthalmic care

Stuart Richer, O.D., Ph.D.,a,b William Stiles, M.D.,a and Carla Thomasa

aEye Clinic, Department of Veterans Affairs Medical Center, North Chicago, Illinois; and bRosalind Franklin University ofMedicine & Science, North Chicago, Illinois.

KEYWORDS Abstract

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RPE lipofuscin;Dietary polyphenols;

Resveratrol

BACKGROUND: Lipofuscin is the most consistent and phylogenically constant morphologic marker ofcellular aging. Autofluorescence of the A2E fluorophore within retinal pigment epithelial (RPE) lipo-fuscin affords the opportunity for noninvasive evaluation of age- and disease-related pathophysiolog-ical changes in the human retina. It is being used in National Eye Institute/Age-Related Eye DiseaseStudy II to evaluate age-related macular degeneration (AMD) geographic atrophy expansion. Experi-ments show lipofuscin can be reversed in cell culture and animal models in heart, brain, spinal cord,and retinal tissues, using an array of antioxidants and iron chelators.METHODS: An 80-year-old man with a gastric resection presented with complaints of unremittingnight driving difficulty despite treatment with lutein and omega III fatty acids. Notable parafoveal de-position of retinal lipofuscin by 50� fundus auto-fluorescence (580 nm excitation/660 barrier filters)and concurrent abnormalities in non-Snellen measures of visual function–Contrast Sensitivity Func-tion, 6.5� large field tritan threshold, 10� threshold visual fields, and deficits in the National Institutesof Health/National Eye Institute Visual Function Questionnaire (VFQ) 25 subjective night driving/mental health subscale questionnaire were obtained. The patient was placed on an over-the-counterdaily oral polyphenolic mixture containing resveratrol and re-evaluated 5 months later.RESULTS: The data reveal improvements in all measures of visual function, subjective improvement invision and mental functioning on the VFQ 25, and visible clearing of RPE lipofuscin.CONCLUSION: To our knowledge, we believe this to be the first reported human clinical case of lip-ofuscin reversal in the human eye correlated with measured clinical and subjective improvement invisual and mental function after nutraceutical intervention.Optometry 2009;80:695-701

Modern medicine is being ushered into molecular med-icine by the mapping of the human genome and thediscovery of molecules that significantly differentiategene expression, a frontier of scientific discovery calledepigenetics. Small molecules have been identified for theirbroad genomic effects because of their ability to enter bothcell membranes and the cell nucleus to ultimately influencegenetic machinery.1,2

Corresponding author: Stuart Richer, O.D., Ph.D., Eye Clinic 112e, De-

tment of Veterans Affairs Medical Center, 3001 Green Bay Road, North

icago, Illinois 60064-3095.

E-mail: Stuart.Richer1@VA.Gov

9-1839/09/$ -see front matter–This is a U.S. government work. There are no r

tometric Association.

:10.1016/j.optm.2009.03.018

Small natural molecules known as polyphenols (i.e.,resveratrol, quercetin, ferulic acid, curcumin, and catechin),commonly found in grapes, onions, berries, and rice bran,are notable examples of widely available over-the-counter(OTC) nutriceuticals. The impact of these molecules ongene expression, with the aim of determining how they in-fluence human health, is a field called nutrigenomics.3

Plants that produce these molecules are subjected to envi-ronmental stress (heat, cold, solar radiation, fungal attack),genetically up-regulating production of these defensivemolecules, which are then passed on to humans when con-sumed in the diet, through a phenomenon called xenohorm-esis.4 Hormesis then is a generally favorable biological

estrictions on its use. Published by Elsevier Inc. on behalf of the American

Figure 1 Autofluorescence 14� macular pigment distribution.

696 Optometry, Vol 80, No 12, December 2009

response produced to low exposures to toxins and otherstressors, such as heat, cold, and sunlight.

Ocular aging and its marker: lipofuscin

Just how genomic medicine will impact the future ofophthalmic care is now being evaluated. In question iswhen to invoke molecular medicine to reverse existingdisease: (1) when the first signs of eye aging begin, such asdeposition of retinal lipofuscin or (2) later in the patho-physiological process? It is possible that some agingchanges can be reversed to some extent, such as lipofuscindeposits, whereas others cannot, such as lost post–mitoticretinal pigment epithelial (RPE) cells.

Lipofuscin is a universal marker of mammalian agingcomposed of cellular debris. The progressive accumulationof lipofuscin has been called a garbage catastrophe.

However, lipofuscin is not a benign marker of aging butrather generates free radicals impairing cellular function-ing.5 The amount of lipofuscin within the cytoplasm of rab-bit cells is minuscule, representing only 0.29% of thecytoplasm volume at 12 months of life, whereas it reaches2% of cytoplasm volume at 79 months (7 times increase).6

Lipofuscin and the aging eye

Electron microscope analysis of tissue extracts of humanRPE, the single-cell wall of phagocytic (digesting cells) thatreside between the blood layer (choriocapillaris and choroid)and photoreceptors (cones, rods), shows no lipofuscin gran-ules in young eyes but large numbers in older eyes.7

Accumulation of cellular debris (lipofuscin) at the back ofthe human eye (retina) does not begin until age 19.8 Retinal lip-ofuscin deposits, induced by iron accumulation and calcifica-tion in retinal tissues, are generally not found until the thirddecade of life, and more advanced cellular lipoproteinaceousdeposits in the retina, called drusen, are not normally observedduring an eye examination until the fifth decade of life.9

In other words, about when full childhood growth ceasesis when lipofuscin begins to accumulate more rapidly. Thisis also precisely when the demand for iron to make newRBCs declines.

Ocular lipofuscin measurement

Unlike other organs of the body, the accumulation of retinallipofuscin with advancing age can be observed directlywith the use of a scanning laser ophthalmoscope orspecialized photographic systems.10 Thus, theoretically,lipofuscin measurements serve as a way to determine thebiological aging of the eye. The human eye, being a trans-parent organ, is subject to bombardment by solar ultraviolet(UV) and blue light radiation that induces oxygen free rad-icals accelerating the accumulation of lipofuscin more sothan other organs. The human eye may age at a fasterrate than the rest of the body. In fact, visual decline is thefirst sign in lipofuscin disorders (lipofuscinosis).11,12

The formation of lipofuscin increases after the removal ofa cataractous (cloudy) natural focusing lens of the eye andimplantation of a clear plastic intraocular lens. The com-bined implantation of a yellow intraocular lens (UV bluefilter) and use of a vermillion sunglass filter would theoret-ically reduce lipofuscin formation.13 The accumulation of

Figure 2 A, B, Significantly improved contrast sensitivity.

Richer et al Clinical Research 697

retinal lipofuscin correlates with declines in contrast sensi-tivity and visual acuity.14

Prior attempts to reduce lipofuscin

Experiments have found that lipofuscin accumulation can beincreased, decreased, or even reversed in cell culture andanimal models, in heart, brain, spinal cord and retinal tissues,using an array of oxidants, antioxidants, and iron chelators,particularly vitamin E, lipoic acid, carnitine, grape seedflavonols, curcumin, coenzyme Q10, and glutathione.15-27

Decades ago, attempts were made to reverse retinalaging and reduce lipofuscin deposits by the use of anacetycholine precursor (DMAE), which produced mixedresults in 2 human studies.28,29 These studies found that atherapeutic intervention in the aging retina is possible.

Iron, retinal lipofuscin and age-relatedmacular degeneration

Lysosomal compartments in mammalian cells produceenzymes that act to scavenge cellular debris, are rich in

labile iron, and are subject to oxidative stress throughFenton-type reactions (forming hydroxyl radicals). Lipidperoxidation, in turn, induces the formation of lipofuscin.30

Old cells have 10-fold more iron content than youngcells.31 As cells age, they accumulate this iron-generatedlipofuscin, which impairs cellular functions. Iron chelatorsare proposed to retard or erase lipofuscin.32

Experimentally injecting iron into the vitreous gel of theeye induces accumulation of inclusions with lipofuscinlikefluorescence in the RPE.33 Age-related macular degeneration(AMD) retinas have more iron within the photoreceptors,RPE, and drusen than do age-matched control retinas. Accel-erated AMD-like maculopathy develops in patients withretinal iron overload from the hereditary disease aceruloplas-minemia (lack of the major copper transport protein). Micewith retinal iron overload resulting from knockout of cerulo-plasmin exhibit retinal degeneration with some features ofAMD. Iron chelation can reduce iron overload in mice andprotect them from degeneration. The prospect of using ironchelators to reduce oxidative stress in the retina has been pro-posed.34 The use of iron chelators has also been shown to re-verse the accumulation of cellular debris within aged cells, in

Figure 3 A, B, Kinetic field vision analysis.

698 Optometry, Vol 80, No 12, December 2009

a similar manner to calorie restriction.35 Iron chelation alsoreverses the progressive rupture of lysosomes and prolongsthe life of cells.36

Calorie restriction

Calorie restriction unequivocally and universally lengthensthe average and maximal life span of all organismsincluding yeast cells, fruit flies, roundworms, fish, rodents,and presumably humans.37

While the accumulation of lipofuscin as an agingpigment is a universal feature of aging in animals, partic-ularly in the RPE of the eye, dietary restriction with 40%fewer calories, has been shown to decrease RPE lipofuscinaccumulation.38 Calorie restriction is known to activateautophagy (cell cleansing). Near starvation also increasesautophagy and longevity.39

A prevalent misunderstanding in antiaging medicine isthe false belief that food-restricted diets exert beneficialeffects via limited calories. Researchers at McGill Univer-sity conclusively showed that calorie restriction limitsmineral intake, which then slows aging as evidenced by arapid increase of aging markers in the brain (when acalorie-restricted diet plus added minerals was comparedwith a calorie-restricted or ad libitum diet).40 Thus, it is notcalories, but rather mineral intake, that appears to drive therate of aging. A low-calorie diet limits intake of major min-erals such as iron, copper, and calcium, which then retardsaging.

Food-deprivation diets as therapy for aging diseases arenot likely to be achievable for socioeconomic reasons. Thishas prompted researchers in the field of aging to search formolecular shortcuts. Once the discovery that calorie re-striction activates the SIR2 gene in lower forms of life,41

homologous with the Sirtuin 1 gene in humans, researcherslaunched a fervent search for molecules that could mimiccalorie restriction via activation of the Sirtuin 1 gene.

Small molecule calorie restriction mimics

It did not take long for investigators in the field of aging toidentify candidate molecules for activation of the Sirtuin1 gene. Small polyphenolic molecules, namely resveratrol,fisetin, and quercetin, have been identified as calorierestriction mimics, activating a key DNA repair gene,Sirtuin 1, that is up-regulated during periods of fooddeprivation. Resveratrol exerts the strongest Sirtuin 1 acti-vation among the polyphenols tested.42

It is not surprising that small molecule therapy isproposed to intervene in the aging process to slow theaccumulation of lipofuscin and the eventual onset of thecommon slow-progressive form of AMD.43 It is interestingto note that resveratrol activates autophagy (cell cleansing),which can either prolong the life of healthy cells or shortenthe life of tumor cells.44

As described previously, metal chelation is indicated forpreventive and therapeutic intervention in age-related ret-inal disease, and small polyphenolic molecules both

Figure 4 A, B, Serial lipofuscin images.

Richer et al Clinical Research 699

activate the Sirtuin 1 gene and are metal chelators. Smallpolyphenolic molecules are also strong metallic metalchelators, with a higher reducing capacity for copper thaniron ions.45

By their ability to chelate minerals, polyphenols altergene expression at very low concentrations.46 Gene arraytechnology shows that iron excess or depletion affects sev-eral hundred genes and that iron chelation is proposed asbeneficially influencing the genome.47 Use of small-mole-cule calorie restriction mimics, which produce their benefi-cial effects via mineral chelation and subsequently exertmeasurable genomic effects, is on the therapeutic drawingboard.

That human populations who consume a reduced-caloriediet (40% fewer calories by residents of Okinawa, 25%fewer calories by women compared with men) only liveanother 4 to 5 years48 could be explained by the realizationthat minerals, and not calories per se, control the rate of ag-ing. The use of iron-chelating polyphenolic molecules hasbeen proposed as an intervention that addresses a widerange of age-related diseases such as Alzheimer’s, Parkin-son’s, cardiovascular, and immune-compromised disease.49

The hope is that via molecular medicine, true preventionvia inhibition of lipofuscin can be realized before loss ofvision, and that chelation of iron and copper from the retinawould achieve visual regeneration among those withalready existing pathologic visual decline. A case presen-tation may serve as an early example.

An 80-year-old white man presented to the Departmentof Veterans Affairs Medical Center Eye Clinic in North

Chicago, Illinois, with need for an updated refraction toreceive his new driver’s license, with the complaint ‘‘I can’tsee at night.’’ Ocular history included early nuclear cataractright eye, pseudophakia (intraocular lens) left eye, aniso-metropia (unequal refractive error), exophoria, and rightpseudoexfoliation with normal intraocular pressures of 15mmHg in both eyes. Additional medical history includes:� 65% gastric resection (1956) with impaired vitamin

B12 processing (B12 shot twice a month), anemia(iron shot every month)

� Past alcohol abuse (1960s)� Bilateral hip replacement� Spinal stenosis of third to fifth vertebrae upper and

lower back� Left carpal tunnel� Large, efficient slow-pumping heart� Familial history of AMD (mother in late 80s)His refraction and Snellen Visual Acuity were:10.75 w 11.50 x 10 1BI ————20/20–1.50 w 1 0.25 x 150 1BI slab off 20/2012.50 add trifocalsThis represented a minimal change in prescription. Slab

off was included in the prescription to reduce opticallyinduced anisometropic vertical phoria. One degree fovealmacular pigment was measured by heterochromic flickerphotometry using a QuantifEye� (ZeaVision, St. Louis,Missouri) device and found to be normal albeit slightlyreduced in the left eye (0.53 density units [du] right 0.24 du1eft). Three-dimensional auto-fluorescence macular pig-ment imaging confirmed the asymmetry (see Figure 1).

700 Optometry, Vol 80, No 12, December 2009

A blue-free 3200 Kelvin low-vision reading lamp,traditional OTC ophthalmic supplements, and new eye-glasses were prescribed. However, his night visual symp-toms were uncharacteristically not responsive to carotenoidand fish oil supplementation. Better than 75% of malepatients in this age group typically respond to thesesupplements in our clinic.50-56

On closer evaluation, we measured abnormalities on the100-point National Institute of Health/National Eye InstituteVisual Function Questionnaire (VFQ25) including NightDriving and Mental Health Subscales, an abnormal area un-der the curve (AUC) contrast sensitivity function (Func-tional Vision Analyzer�; Stereo Optical Company, Inc.,Chicago, Illinois) (see Figure 2A), abnormal 6.5� (largefield) color vision disturbances (ChromaTest�; CH Elec-tronics, Bromley, United Kingdom), and abnormalities inhis central macular 10� kinetic macular visual fields at 5 dis-crete contrast levels (SimulEyes Kinetic Macula FieldTest�; Rush Ophthalmics, Gold Beach, Oregon) (seeFigure 3A). There was also notable parafoveal depositionof retinal lipofuscin by 50� fundus autofluorescence (KowaMedical Instruments, JP modified Fundus Camera, 580 nmexcitation/660 barrier filters, high output flash, and highgain Hamamatsu C9440-05G digital black and white camera;Kowa Medical products, Tokyo, Japan) (see Figure 4A).

An OTC mitochondrial-support dietary supplement pro-viding resveratrol (Longevinex�; Reservatol Partners,LLC, Las Vegas, Nevada), quercetin, rice bran IP6, andlecithin was prescribed on September 6, 2007. A 5-monthfollow-up visit on February 13, 2008, found improvementin all subjective visual function parameters and VFQ 25night driving and mental health subscales.� Visual acuity 20/20 1 3 right (better) 20/20 1 1 left

(same)� VFQ25 5 84 versus 81 Overall Scoredimproved

� 83 versus 67 Driving Subscale Scoredimproved� 100 versus 80 Mental Health Subscale Scoreddra-

matic improvement� StereoOptical Contrast Sensitivity Function:

376 AUC right 533 AUC leftdmuch improved (seeFigure 2B)

� 6.5� Tritan Chroma� test, 8.6 SD right; 5.5 SDleftdbetter

� 10� Kinetic Macular Visual Fieldsdmuch improvedright; normalized left (see Figure 3B)

These dramatic subjective results were supported byobjective clearing of retinal lipofuscin (see Figure 4B) andimprovement in self-reported night vision and cognitiveability as well as dramatic improvement in contrast sensi-tivity function (both eyes) by objective testing when anOTC dietary supplement (Longevinex�), providing a blendof proprietary nutriceuticals (resveratrol, quercetin, ricebran phytate) was added to the patient’s dietary supplementregimen. Subjective response: ‘‘My night vision and think-ing have gotten much better.’’

We remain cognizant to the possibility that the originalsupplements were continued over the 5-month polyphenol

intervention and may have had a salutary effect. Thisadditionally argues that a detailed assessment of nutriceut-icals should be ascertained before adding another dietarysupplement. Nonetheless, this case may serve as an earlyexample of the potential for molecular medicine to make animpact in ophthalmic care. To our knowledge, we believe itto be the first reported human clinical case of lipofuscinreversal in the human eye correlated with measured clinicaland subjective improvement in visual and mental functionafter nutriceutical intervention. Furthermore, it becomesevident that by measuring lipofuscin deposits, not only canthe biological age of the human eye be assessed apart fromits chronological (calendar) age, but more significantly,such measurement may serve to help determine the bio-logical age of the entire body. Pharmacologic and nutra-ceutical systemwide age-reversing effects could then beestimated in this manner.

Acknowledgements

This material is based upon work supported by the DVAMedical Center, North Chicago, Illinois and the Departmentof Veteran’s Affairs. The authors thank Kowa Optimed USA(Torrance, California, and Japan Corporate Headquarters);Rush Instruments (Gold Beach, Oregon); Stereo Optical(Chicago, Illinois), and Zeavision (St. Louis, Missouri) fordonation of ophthalmic equipment. The OTC dietary sup-plement (Logevinex�) was donated by the manufacturer,Resveratrol Partners LLC (Las Vegas, Nevada).

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