What's up Okay, what's that? Okay, thank you very much. It's nice to give kind of an overview of some of my long-term interests in terms of Treatment and biochemistry of retinal diseases, and we're gonna be focusing today on phytochemicals and Just Would that drop out? Okay, so phytochemicals are plant derived natural products And as some of you may know who's ever been to my house that I have a long-term interest in plants the specifically Exotic trees and shown here are just a few Examples of some of the trees. These are not the actual trees, but are the Web versions of these are some of the rarer trees that I have in my collection at home I have somewhere over 50 different rare trees on a very relatively small plot of land and The the rarest tree I have are the the most uncommon tree of all that I have is called a willy me pine Which is shown here, which was only discovered in Australia in 1993. They do grow over a hundred feet tall And I have a very nice specimen, although it's not nearly as big as this one a small specimen here There are this is a cryptomoria japanica, which is a Japanese tree This is found in the imperial gardens if you ever go to Japan This one I particularly like called the blue ice Arizona Cyprus. It's got a very kind of scaly needles and it kind of and But is again it grows it's very hardy for this type of climate here And then the most the rarest tree I have of all is called a chihuahua spruce Which looks just like a blue spruce, but it's found in only a very remote part of Mexico, and I bought this From a from an unusual source online, but I was able to get this one here It's main claim to fame is it looks like a blue spruce, but it's much much spikier So deer won't eat it. It's the big thing on this and as far as I know I have the only specimen of this in the in all of Utah In fact, I've only seen it one other time anywhere in the United States once in San Diego so Anyway, but this to put this in perspective My tree collecting passion is one reason why I'm even here today When I put this down when I put down on my college and my medical school applications that I was a tree collector That caught the interviewer's eyes every time I would ever had an interview I could spend half the time on the interview talking about that rather than science But I think it definitely distinguished me when I was applying to all to medical school so anyway, there is certainly a long history of Plant-derived chemicals that are important in the eye and we don't have time to go through the whole big ocular disease talk but clearly there are a number of Compounds that we use Commonly every day that our plant-derived things such as Pylocarpene and atropine to dilate pupils come from the pharmacopoeia going back hundreds of years cyclosporin a although is derived from a fungus and that's something we use for dry eye and But we're going to be focusing today on retina which certainly has a long phytochemical disease of phytochemicals so and they're multiple They're multiple Conditions where they may play an important role in the in the eye and these include night blindness retinal degenerations Bacular dystrophies nutritional maculopathy is an age-related macular degeneration So phytochemicals have been known for a long long time going back as far as some of the ancient papyri of Egypt Where it was known that vitamin aid that vitamin a type compounds either liver or other plant-derived compounds That were somehow rich in vitamin a was known to the ancient Egyptians to be good for treating night blindness And vitamin a deficiency is certainly a common disease in the developing world It's rare here in this world in this in the developed world And although we have seen a few patients with this with associated with malabsorption syndromes the night blindness typically precedes the ocular surface disease and it has to do with the fact that that retinoid A compounds are key for the visual cycle that we see in the eye and this was Related to some of the work that I did my PhD on of the isomerization of all trans retinoids to 11 cis retinoids is the key step in the visual cycle that occurs in the retinal pigment epithelium and Without enough vitamin aid to drive this part of this process we develop night blindness and eventually you can get an unusual form of Retinopathy and shown here is it as an example of the kind of thing you may see an extreme vitamin aid deficiency where you get yellowish white dots in the periphery and this kind of patient would be Would have very narrow visual fields almost an RP type syndrome of night blindness and very narrow narrow fields And if you can just re recharge the vitamin a cycle you will see Good response in these patients Phytochemicals because of this phytochemicals have been looked at for retinal degenerations and retinitis pigmentosa, which is shown here Has been related to some defects in these visual fields in the visual cycle and so it was Proposed by Burson about 20 years ago that vitamin a supplementation might be helpful in some RP patients and he conducted some large-scale Large-scale Goal trials where he used vitamin a supplementation at a relatively high dose 15,000 units per day in the retinal palmitate form and he did show that there was a small but subtle Decrease in the progression of retinitis pigmentosa now this is Kind of we now know that retinitis pigmentosa is really a group of 30 At least 30 different mutations, so it's really many many different diseases going on So it's not obvious that vitamin a should really make a difference But it still is part of clinical practice and many retinol specialties to do to give vitamin a supplementation And I offer it to my patients But I also offer to tell them that they have to be to have very modest modest Expectations for any response to this Because it's just a decrease in the progression of the loss of the cone cone erg So and you also have to be careful, of course, because vitamin a has its own toxicities It can it's teratogenic in women of childbearing age at least at this dose And it's also potentially can cause liver function abnormalities in these patients He's also done further phytochemical studies in the last decade or so He's also looked at omega-3 fatty acid supplementation Which also seemed to help but the effect was even weaker and within the past year He also looked at lutein supplementation will be covering carotenoids in much more detail later on in this talk And that was also a very weak effect It did not reach its primary endpoint was only on subsequent analysis that they found Some decrease in progression of loss of visual field in the periphery in these pay in the far periphery in these patients and Based on some work that we did earlier on where we measured about 10 years ago where we measured Lutein levels the macular pigment levels in the eye and RP patients We found that there is that they don't have any abnormalities. They're actually normal to high So it's not obvious that this was really even a great great way to approach RP And of course phytochemicals can make some forms of retinitis pigmentosa worse Disease is a very uncommon disease But that has to do it's it gives you a retinal degeneration and that has to do with an inability to metabolize phytanic acid which is an unusual branched chain fatty acids saturated fatty acid here and Patients who have who are diagnosed with refsome disease Need to have a diet low in phytol of the precursor phytol and phytanic acid Which means no glee green leafy vegetables no animal fats and no milk products. So what's left? It's pretty much just cereals. So it's a pretty pretty nasty disease to have in terms of treatment and Then the other one that famously shows up on boards and on questions is gyrate atrophy again Both of these are so rare that I've never actually seen a patient with either of these But that's a defect in ornithine metabolism a An amino acid here that's uncommonly in the diet and that's treated with a low protein low arginine diet Phytochemicals also can play a role in macular dystrophies that we most commonly see a stargardt disease which You know affects about one to about one twenty five thousand people are affected in the United States forms the Stargardt these which accounts for about 95 percent of the cases and is caused by a defect in the ABCA 4 gene and the dominant form which is much less common But we have a very interesting family here in Utah with this disease that accounts That's caused by a defect in the ELOvl 4 gene and both of these have nutritional interventions that potentially can be important for them For Stargardt one Stargardt one is a defect in the ABCA for protein Which transports excess vitamin a aldehyde out of the photoreceptor segments as part of the visual cycle Now we talked about the importance of vitamin a in Normal vision, but too much of it can cause a lot of problems and if it sticks around too long in the in the photoreceptors before it goes to the RPE to be reisomerized It can start having reactions with the phosphatidylethanolamine and create a toxic compound here Called a2e which is here, and there are also a lot of other bis retinoids that Conform with these reactive aldehydes the vitamin a and these are these generate free radicals They also seem to disrupt a lot of enzymatic processes in the eye, and they're very very indigestible And they're part of the lipophusin that is a component of malfunction of the RPE So the thought with Stargardt one is that it may actually be good to restrict the vitamin a in the in these patients And so there are interventions of various medications or even dietary interventions that we recommend for the patients that To try to keep down the levels of vitamin a to decrease the formation of this a2e And those are still in progress, but there are some clinical trials that will be occurring in the foreseeable future Stargardt 3 is another one that we think may be very important and have nutritional interventions Stargardt 3 is a defect in the ELOVL4 gene which is involved in the very complicated pathways of synthesis of omega-3 and omega-6 fatty acids in the eye and in In animals and in humans that have the ELOVL4 defect They don't make enough of a lot of the omega-3 omega-6's Perhaps EPA and DHA and not shown here They definitely do not make some of the very long chain fatty acids that are metabolites of EPA and DHA Going on to the C30 up to C36 So the thought with this is that somehow you need to get more of these very long chain fatty acids Into patients and so we have a clinical trial right now on our patients on our family with ELOVL4 Mutations to give them to encourage them to consume more fish and EPA and DHA either through algae sources or through Or through fish consumption to see if we can slow down the progression of the disease This is an open label small trial There's not enough people to ever do an randomized trial on this But this is in progress and for the residents who are with me occasionally. You'll see these patients coming through And of course if phytochemicals can be helpful in some patients they can also make things worse Shown here a couple of classic examples can't the xanthan which is a carotenoid used for skin tanning can can crystallize in the eye and Although I don't know why this keeps dropping out, but it does Although visual loss is rare so can't the xanthan is Has been described well described in the literature you get this golden crystal retinopathy that occurs in the eye And so can't the xanthan is certainly not recommended in high doses It's still in our food supply as a food colorant and it's still available on the internet actually a pretty high doses Although I haven't seen anyone coming in looking like this Other ones that are of other phytochemicals that are of concern to the retina include things like oxalic acid Which can crystallize in the retina and nicotinic acid which can cause cystoid macular edema at high doses And that's still commonly used to treat high cholesterol But enough of that we're gonna be focusing on age-related macular degeneration and Which is certainly the focus of a lot of my clinical practice and where phytochemicals definitely can play an important role And as we all know age-related macular degeneration is the leading cause or up until recently at least I'm not sure it's still true anymore is the leading cause of irreversible vision loss in developed countries Somewhere between 1.7 and 20 million Americans have age-related macular degeneration Depending on how you define it and they're about 200 there 200,000 new advanced cases every year It's a disease that increases whose prevalence increases dramatically with age and it comes in both the wet and the dry form The wet form accounts for a smaller portion of AMD, but that's certainly been the focus of a lot of our treatment And up until the day the days that we started getting anti-veg F compounds it accounted for about 90% of the blindness and clearly it's an important public health problem the The rate especially in whites rises dramatically as we as we age We probably will never completely eliminate age-related macular degeneration, but just if we can Delay it somewhat and move the curve to the right more we can certainly we can improve quality of life for many many of our patients and that's why prevention is important and Just shown here are some classic pictures of age-related macular degeneration both in the wet form and in the dry form that in The end end up in a common pathway with severe scarring and loss of central vision We've developed great new treatments for age-related macular degeneration in my time here on the faculty It's gone from a disease where we really had no options for the patients We would tell them you've lost vision Maybe we can do some laser and you'd probably even get worse But in the long term you're better off to a disease. That's that's certainly manageable We do lots and lots of injections into the eye. We now have Very different type of patients. We have happy AMD patients who are at least seen better They're not happy about coming in perhaps monthly for Lucentus injections, but at least their vision is much better and They don't have they don't have to deal with the horrible natural history of this But with all of these treatments, they're expensive. They're a burden to both the doctors and to the patients So prevention is important so if you're going to prevent a disease you have to look at at What are the various risk factors for this and how can we modify them certainly? There is a large group of non modifiable risk factors These include age which of course is an important risk factor We've learned over the last decade that heredity is a very very important risk factor Probably accounting for well over well over half of the risk of even getting age age related macular degeneration Gender is a more minor risk factor. It seems to be more prevalent in women But there's also many more elderly women that we're seeing and it does seem to be more common in lightly pigmented people So in lightly pigmented races, but for the but these are not particularly modifiable We really have to focus on the modifiable risk factors We know that smoking is very very important as a risk factor That's shown up in multiple epidemiology studies and needs and always needs to be addressed with any of our patients Who are smokers it appears to go with cardiovascular risk factors blood lipid status and hypertension So those need to be treated. It's a little more controversial about alcohol consumption But it appears to be at least low low alcohol consumption just like it is for cardiovascular disease Maybe protective in some in some patients light exposure at high levels probably is a risk factor Although it's controversial at the more normal ambient Light levels that we're exposed to but we'll be focusing today on nutrition And so why should this be important? Well the retina and the retinal pigment epithelium have very highly unsaturated lipids and these are susceptible to oxidative damage And this is and the macula itself is an area that's exposed to high high levels of oxygen and high levels of light almost by definition so if we accept the premise that Age-related macular degeneration is at least in part a disease of oxidative stress It makes sense that antioxidant nutrients which are the most common antioxidants We deal with naturally might play a role in protection against age-related macular degeneration But as we'll see these are difficult studies to perform age-related macular degeneration is a complex disease Nutrition is a complex factor putting into this plus we have all the other factors that have that are involved in progression of age-related macular degeneration on the other hand nutrition is Something that everyone can participate in it's not expensive and it's something that That most patients with encouragement can modify So to identify Nutritional factors that might be associated with age-related macular degeneration They have to there are many different ways we can study this the classic way that's been done probably for the last few decades Is epidemiology looking at large populations looking at who gets age-related macular degeneration and who doesn't and who gets and What are what kind of nutrients have they been been consuming or other risk other? environmental risk factors like focus like smoking and from that they can derive at least good Hypotheses of what needs to be tested in a more prospective manner. These will not give you cause and effect But at least they can give you a lot of information Animal studies are a little more difficult because animals Don't get the same type of macular don't get macular degeneration. They don't have maculas and they may even Metabolize differently from what we what we do just because we absorb a metabolite very very well Doesn't mean it's going to work the same in an animal model like a mouse and this is particularly true Say for carotenoids my area of research We as humans take up carotenoids very readily from the diet and deposited in our tissues in mice You can give massive doses many much more than I would even administer to a human and they just don't even pick it up They just They their body doesn't absorb it. They don't deposit it in their eye So you can't look at right now You can't look at mouse models of retinal disease and really and just try to feed them carotenoids and see and hope that you're Going to learn a lot about the human condition My laboratory across the way is dedicated to physiology and biochemistry of trying to understand these nutritional factors and Among some of the key things that you want to see are that Nutrients should be found in appropriate quantities of the retina in the retina So we work with autopsy eyes and we actually measure various nutrients in the eye if you're going to say a nutrients important You better be able to see it in the eye Otherwise, it's probably not all that relevant. They should have physiologically plausible mechanisms So if you say something's working well as an antioxidant or a light screening compound you should be able to show that in vitro and Finally, there should be some evidence that deficiency states might be associated with higher risk of age-related macular degeneration And eventually you have to do some sort of prospective trials and I'll cover some of this Obviously, this is way too much to cover in just one talk But we'll talk about some of our perspectives of what we've been finding on the physiology and biochemistry of some of these compounds So there is a rich epidemiology literature and over the years a number of nutrients have been Epidemiologically linked to decreased age-related macular degeneration risk and these range from antioxidant minerals such as zinc and selenium and some of the original zinc studies were done by our former faculty member Mano Swartz about 20 years ago here at University of Utah and these antioxidant minerals are cofactors for some of the antioxidant enzymes in the eye and It was thought found that taking high doses of this at least epidemiologically and in small clinical trials Seem to be protective and this has been tested in further trials Antioxidant vitamins such as vitamin C E and A are good antioxidants that have been tried in number of trials and are certainly linked epidemiologically Polyunsaturated fats like DHA EPA and their precursors Have been shown to be important and that's being part of tested now in arids to study I'll be covering today the my area of primary interest Which is lutein and zeaxanthin the carotenoids that make up the macular pigment and to a lesser extent other carotenoids have also been linked Epidemiologically such as beta carotene and lycopene, which is beta carotene found in carrots Lutein and zeaxanthin are found in green and yellow fruits and vegetables and lycopene which is found in tomatoes Some of these beta carotene and lycopene are not nearly as strong And they don't fulfill one of the main criteria of being found in large amounts in the eye Almost none is found in the eye, so they're not they haven't been an area of focus quite as much And then there are herbals which many of our patients talk about all the time things like billberry Resveratrol other things like that and I'll touch upon these towards the end There's obviously not nearly as much information on them So our current Treatments for age-related macular degeneration and prevention is based on the age-related eye disease study The first one that was done and they used the the nutritional knowledge of the 1980s basically to come up with a supplementation regimen that they tested on nearly 5,000 subjects Ranging in the age certainly in the amd age and follow them for five years So this is a good high quality study that was sufficiently powered and they were randomized to antioxidant supplementation But they didn't know anything about lutein and zeaxanthin which we'll talk about later So those weren't part of it and they looked at the incidents of cataracts severe vision loss and age-related macular degeneration progression and They had to because it was a clinical trial They had to certainly grade all of the patients to put them into the right categories And they had categories one through four which are shown here And they found fairly early on that they really shouldn't be looking at grades one and grade two These are essentially the worried well that we deal with in our clinic They have maybe a few drusen or a few small drusen or some pigmentary abnormalities But natural history of these patients is so slow I mean the risk of developing age-related macular degeneration in five years is one percent or less probably in this sort of Category so it's not worth studying these patients You really want to look at the higher risk ones in a clinical trial like the a reds to a reds one study So they either had to have extensive intermediate drusen or large drusen or non-central atrophy But still had good acuity something that could be Where you could follow these patients or they had to have No, or they could have advanced age-related macular degeneration in the fellow. I and And a certain visual but good visual acuity and milder AMD in the good eye The a reds formulation was based on what as I said the nutritional knowledge of the 1980s So they took 80 milligrams of zinc oxide. This is really a pretty high dose And not a very bio available form of zinc, but that's what they chose This dose of zinc is so high that it can cause anemia that and that's why they added had to add two milligrams of copper oxide To prevent the anemia that would be caused they had a reasonable moderate dose of 500 milligrams of vitamin C and 400 international units of vitamin E and they had they chose a very high dose of beta carotene 15 milligrams per day, which is 25,000 international units. They did this, you know beta carotene is not Even though it's not found high in the eye. It's metabolites. It's broken down to retina to retinoids So it was a reasonable choice for this, but in retrospect They found this was too high at too high a level to be to be Using but this was available at the time in 1980s was available in in large quantities And they followed these patients for five to seven years And they found actually to a lot of clinicians surprised that the antioxidant intervention really did make a difference Shown here is the placebo line here for risk of progression to severe vision loss And the patients who were randomized to the combination of antioxidants and zinc did the best There was a reduction of about 25 percent of the rate of progression over the five to seven years here and it was less if you took just antioxidants or zinc itself and This is real, but it's relatively modest But when you think about a disease that potentially affects millions of people this This 25% risk reduction really is important and that's why it was rapidly Translated into clinical practice and recommendations and this was not due to progression of cataract at least in the United States Population there was no difference between patients getting placebo or not And it was all driven by primarily risk of developing advanced age-related macular degeneration either geographic atrophy or Coroidal neovascularization and again it was the same that the patients did best if they were on antioxidants and zinc so the summary of what a reds one taught us is that there could be a That in a dietary intervention or a nutritional supplement intervention could cause could result in a significant Reduction in progression of age-related macular degeneration at least in the high-risk people It's harder to translate the the a reds pop recommendation again to the worried well the patients who are have a family history who are 40 or 50 years old We at least I still tend to recommend diet rather than taking high-dose supplements in those patients This did not seem to have much for reduction in cataract progression, but clearly this is just the first generation Supplementation it's kind of like chemotherapy in the 1950s, you know You could prolong life a little bit, but if you really want to get a significant a Really substantial life impacting change you're going to have to look at different combinations You're going to have to try to improve upon the formulation So specifically in the original publication in 2001 They didn't mention that lutein and zeaxanth and some of the more and and also omega-3 fatty acids the newer nutritional knowledge needed to be applied and looked at in optimizing the formulation So a reds 2 was initiated about three or four years ago And we're one of the a reds 2 sites and they haven't we're now studying a new generation formulation Where we're modified where we're adding fish oil and I'll talk a little bit more in some future slides how this was come up Wow, they came up with 1,000 milligrams of EPA and DHA and They added lutein at 10 milligrams and zeaxanthin at 2 milligrams And they also realized that they needed to decrease the levels of zinc because they were having urinary tract Problems in some of the patients and they also needed to reduce the levels of beta carotene Not only might it interfere with uptake of lutein and zeaxanthin But the doses were so high that it was contraindicated in smokers It was found during the a reds 1 study that a number of publications came out that that high dose of beta carotene Increases the risk of cancer in smokers and and although not here in Utah many many AMD patients are smokers. So you're that's not the way you want to go Right now we're studying their 4,000 patients that are more than halfway through the five-year process at 100 sites They're all in their age range of 50 to 80 and they have high-risk dry age related macular degeneration And recruitment is complete, but we still have a number of patients going through So I can't comment yet on what a reds 2 is going to show us It's not going to be published until 2013 is probably when we will have the results But certainly they picked a fairly high-risk population We're getting plenty of progression of the disease in certain in our pot in our patients either to choroidal neovascularization and Or geographic atrophies. So They did choose a good population to study from an epidemi from a clinical trials standpoint, so Why did they put in fish oil? Well? Multiple epidemiology studies have shown we're starting to show through the years that consumption of fish particular in particular fatty fish such as salmon and tuna Had were associated with protection against age-related macular degeneration typically we're talking about reasonable Consumptions two or three servings per week, which for many many patients There are plenty of patients that don't eat fish at all, but they found that Consumption of that level seemed to be associated in in epidemiology with decreased risk of age-related macular degeneration and Physiologically it meets The criteria it's found these omega-3 fatty acids like EPA or especially DHA Constituted up to 30 30 to 40 percent of the photoreceptor the lipids in the photoreceptor membranes So they're found in high amounts low levels of this. We recently published a paper looking at autopsy eyes of of AMD patients versus age match controls and there is about a 30 20 to 30 percent reduction in Omega-3 fatty acid levels in patients with with early AMD in the in the autopsy eyes And it's also been shown protective as we as we mentioned before in a reasonable model for dry macular degeneration Star guard 3 and also as a as a nutritional intervention. It is safe and well tolerated omega-3 fish is Does not have side effects as far as we too many side effects to people and the omega-3 fatty acids that you can get in Pilform at the 1,000 milligram level are pretty safe The main problem is that patients don't like the fishy taste that they sometimes get but in terms of bleeding other problems There is there. It's a very safe dose the high doses that cardiovascular Physicians use up to up to three grams per day can cause bleeding problems, but it's That it's not found at the 1,000 milligram level and We in my laboratory are studying a lot of the metabolites these very long-chain Fatty acids that are metabolites, but I don't have time today to cover that We're going to be focusing primarily on the xanthophils the carotenoids of the macular pigment so I've been studying the macular pigment now for 15 or 20 years I think that the macular pigment is the yellow Pigmentation right at the fovea and this is a cross section a classic cross section of a monkey fovea showing that's unstained Just showing how yellow the macula is and for those of you who know some history or Latin The macula actually stands for the macula lutea or the yellow spot This has been known going back to the 1700s that the macula of the primate eye is Very different very unusual it has this yellow spot and one of the reasons I got into the macular pigment field is Looking at this and wondering biochemically how and why this is occurring Why is this so important and why does the body go the human and body go out of its way to concentrate these carotenoids that are found in? specific fruits and vegetables that we consume they're concentrated directly into the macula of the eye and this is found essentially in every eye except in very very unusual Situations and all of the lutein and zeaxanthin that's found here has to come from our diet We cannot make it. We're not like plants that can synthesize lutein and zeaxanthin We can't even make it from closely related carotenoids such as beta carotene It all has to come from the diet and there has been Multiple lines of evidence that indicate from epidemiology that they may be protective Against age-related macular degeneration and there's even some new information coming through that they may be important in infant macular Development we're doing some studies on that So the knowledge of the age of the importance of lutein and zeaxanthin came out during my fellowship from the eye disease case control study 1993 and 94 and they found there was a highly significant inverse correlation between the levels of serum Carotenoid levels and risk of age-related macular degeneration and when they did a sub when Joanna Sudden at Mass Eye and Ear did a subsequent analysis She found that dietary consumption specifically of green leafy vegetables high in lutein and zeaxanthin Looking at this same population were associated with a lower risk of age-related macular degeneration This is an important one this shows the how Epidemiology may be important, but it no one really had ever looked to see if ocular levels of carotenoids are associated with this and We developed just to kind of summarize some work that we did We've been and a number of other people have a number of other centers have developed ways of actually measuring ocular carotenoid levels Inpatients noninvasively so we don't have to take their eyes out and do HPLC on them and there is growing growing Information and growing body of knowledge that ocular carotenoid levels low levels are associated with higher risk of progression to age Related macular degeneration higher levels seem to be protective and so we do offer as a test that as a not as a No-charge test in my clinic various ways of measuring ocular carotenoid levels to see if they can and to see if that To give feedback to our patients whether they're getting enough lutein and zeaxanthin But I'm going to be concentrated today mainly more on biochemistry in the eye and the carotenoids Well, why are carotenoids important? We've already learned they're found at high levels in the eye There's good epidemiology to show they should be studied. What do we know about some of the basic biochemistry? Well, carotenoids are yellow compounds like shown in these yellow peppers here And so they absorb blue light very very well They're yellow filters and they're found in the inner retina before the light hits the most the more sensitive photoreceptors So they're ideal. They're ideally placed to act as an optical filter in the eye There are some animal studies that are on monkeys raised in Oregon on carotenoid free diets That have just been recently published that show that they are more susceptible to light damage Also, they are starting these animals who have been on carotenoid free diets for I think now 15 to 20 years are beginning to show signs of geographic atrophies So they are starting to show signs not only do they get drusen, but they're also starting to at an earlier age They're starting to show at least some signs reminiscent of dry age related macular degeneration We know that the carotenoids in the eye can be manipulated by diet and by supplementation This is a process that however is very very slow. They this To change the levels in macular pigment You have to take the supplements probably for many many months if not a year or two to see To see significant changes within the eye and to cause depletion as they've done in monkeys You probably have to put someone on a carotenoid deficient diet for several years Biochemically they are antioxidants and as we've talked before the retina is exposed to high levels of light in oxygen They can generate free radicals and this can damage photoreceptor Membranes in vitro. They're very efficient quenches of singlet oxygen related free radicals But they're in the inner retina, which is really not the area We think is the highest levels of oxidative stress. So this is still a controversial theory in the field I mentioned before that Carotenoid intake and macular pigment seem to be associated with risk of macular degeneration and can be manipulated My colleagues in Florida Richard Bone and John Landrum did human autopsy studies looking at AMD eyes and age-matched eyes and Found that there's about a 30% lower level of Macular pigment in eyes with age-related macular degeneration and we confirmed this looking at relatively early stage patients using resonance Raman spectroscopy that again this 30% level is very consistently found in patients before the lutein Supplementation era it's getting very hard to do this now that so many of our patients are taking supplements already Dot these we already discussed about supplementation and the macaque studies So what do we do in my laboratory with carotenoids? Well, we're involved in the a reds to study So we're on the clinical side over here in this building where we have about 60 patients involved in the a reds to study We have a sub study where we are doing macular pigment measurements over in research park To follow these patients and we do blood level levels blood level measurements of IH PLC in my laboratory and In associate an association with the physics department We've been developing non-invasive methods to measure macular carotenoid levels both in the eye and in the skin But the focus that I'm going to talk about today is on pure biochemistry I'm going to talk a little bit about how we have identified and characterized How carotenoids even get into the eye so the and this involves binding proteins and from this from the basic biochemistry We hope to uniquely offer Insights into the mechanisms of how carotenoids may protect against age-related macular generation We're doing good on time so Going more into the the basic Biochemistry side we've talked about carotenoids while their carotenoids are the plant pigments in that are universally found in nature and there's somewhere depending on how you count them There's at least 600 different carotenoids in nature. Probably the number is closer to 850 of what is what we really know But we don't eat everything that there is out there so we consume about 50 carotenoids in our diet and Again, these are the ones that are responsible for a lot of the coloration of all of the fruits and vegetables that we eat Not everything in shown here, but an awful lot of it So we consume about 50 of these But there's a level of specificity of these 50 only about 20 of them actually make it into our serum and are detectable if I did a blood sample on you and Of these 20 carotenoids that we consume Only two Lutein and zeaxanthin found in the eye and these aren't even the ones that are found in the highest amounts in our in our bloodstream The highest amounts are things like beta cryptozanthin beta keratin and lycopene are the ones that are found highest amounts So clearly there's a level of selection to bring these compounds into the retina and Even within the macula it's or within the retina itself. There's even more specificity so the macular pigment right here in the fovea is a mixture of Three of these different compounds here some of which are from the diet and some of which are metabolites So we see lutein there. We see it's metabolite three prime epilutein We see zeaxanthin, which is the other one that we get from our diet now We consume in an American diet about two milligrams of lutein Per day per person zeaxanthin is much less common. We consume about zero point two milligrams per day But very unusually there's a lot of mesozeaxanthin, which we never consume in our diet unless you have a diet high in fish scales and That's and shrimps and shrimp shells are good sources of these Most of us don't consume this but in the eye right in the fovea There's a one-to-one-to-one ratio of lutein to zeaxanthin to mesozeaxanthin So there's actually metabolize that metabolism and we and others have shown that lutein is the precursor for mesozeaxanthin And then there's some oxidized compounds here, but the concentration is very high It's greater than one millimolar which is pretty high for a biological compound in a tissue But if you move just a couple millimeters away just to you know Just a little ways into the peripheral macula the concentration will drop by a hundred fold and The ratios change so in the blood and liver our levels are lutein is three to one to zero for lutein to Zeaxanthin to mesozeaxanthin and peripheral retina. There's kind of an intermediate one, but in the macula It's one to one to one So anytime there's a specificity Exquisite biochemical Specificity such as this it's usually mediated by some sort of protein because proteins have are well known to bind hydrophobic molecules like lutein and zeaxanthin and They provide specificity to actually select out the carotenoids that are important and these have been very well characterized And the carotenoid and xanthfil binding proteins have been very well characterized in plants Micro-organisms and invertebrates the most famous one is the one called crustocyanin Which is the responsible for the color of a lobster's shell and this binds astaxanthin a different carotenoid that some people take for AMD but is not found in the eye and that gives their characteristic color here if you boil your lobster you denature the protein it Releases the carotenoid and that's why you see their color change to the bright red here But what kind of proteins are available to humans? Well when I started working this all that was known is that high-density lipoprotein LDL albumin and beta lactic globulin Are involved in mammalian carotenoid? Transport was known that they were the carriers within the bloodstream, but there was absolutely nothing known about carotenoid binding binding proteins in the eye And what could these be doing well? Certainly the most important hypothesis that we're working on is that they could be involved in the selective uptake and Concentration of lutein and zeaxanthin into the macula And once they're into the macula they're very very stable the macular pigment in an autopsy eye stays where it is It doesn't diffuse away it If you put a person on a carotenoid restricted diet It will take years for them to get to lose their carotenoids So they're very very stable in the eat wherever they are in the cytosol the cell membrane or in the cytoskeleton They could be important enzymes Involved in the interconversion of lutein to mesozeaxanthin so that's something we're still looking for and they may facilitate the antioxidant and Protective action of these carotenoids so We spent five to ten years trying to find a protein that in the eye that was responsible for the uptake of Lutein or zeaxanthin into the eye and I also our first important paper came out on this or the most important paper Was the first the identification of gstp1 as the zeaxanthin binding protein in the macula of the human eye This was truly brute-force biochemistry. We got hundreds and hundreds of Maculas from the i-bank here We would grind them up right run them on columns and Follow the carotenoids and follow which proteins were involved were associated with these carotenoids before we could identify That gstp1 a somewhat unexpected protein was the zeaxanthin binding protein And to just summarize quite a few years of work I'll just show you some of the important things that we found is not only was gstp1 purified from the eye associated with the zeaxanthin But it actually did bind zeaxanthin at high levels and shown here a classical binding experiments where we would take Zeaxanthin and purified gstp1 We'd incubate it with this and look at how much would specifically bind and compare that to other proteins that had been theorized To interact with zeaxanthin things like tubulin albumin and a lot of the serum carriers And we could see that very reproducibly zeaxanthin and mesozeaxanthin had a high affinity for gstp1 in the in the sub-micromolar range and That lutein on the other hand which is shown here with the black boxes here Had did not bind to gstp1 So there was selectivity even between zeaxanthin and lutein in the eye in with this protein and then of course you have to be able to show that it's in the right tissues and We took an antibody to gstp1 and we used it to stain a monkey retina here right in the fovea It's because of different fixation. It doesn't look quite as pretty as Max Snotterly's localization of where the carotenoids are the yellow pigmentation But we could see as expected that the zeaxanthin binding protein the gstp1 was concentrated in the Henley fiber layer Right in the foveal pit and was more highly expressed in the fovea than in the peripheral retina. So we solved of One problem we now know at least some way that zeaxanthin was getting into the eye The question was what is the lutein binding protein and we could do our same brute force biochemistry and show that there was a protein Present in the in the human macula that could specifically bind lutein shown here, but we ran into a brick wall We could not identify this protein we would get a reasonably pure preparation And then we would send it off for sequencing and we would get back garbage sequences that didn't of proteins that didn't bind lutein So how are we going to solve this? well, I had to get creative and I Was at a meeting on the international carotenoid society, which is not a Not an eye meeting at all, but it's a gathering that happens every three years of 350 carotenoid scientists So it's botanists plant physiologists everyone anyone who studies carotenoids and we're hoping to have this meeting here in Utah in 2014, but I ran into someone to a plant to an insect physiologist who is studying Lutein binding proteins in silkworms So silkworms eat a lot of mulberry leaves which are high in lutein and this is shown their larvae here And they spin their cocoons and it's well known to the Japanese that you can have different strains of these Of these silkworms some would make white cocoons some would make yellow cocoons, and it was known that the yellow cocoons were were rich in lutein the coloration was covering coming from the lutein there and There the genetics was all worked out And they knew that there were certain mutations and which and it was found that a protein called CBP is responsible for the uptake of lutein from the gut and delivery to the silk plant and CBP is a star D lutein binding protein star D stands for steroidogenic acute regulatory domain protein So just on a hunch I said well, you know obviously even though silkworms and humans don't share a lot of a Lot of structural or any other similarities, but they do have this interest in accumulating lutein Wood is the somehow could this give us a clue as to what the lutein binding protein is so my colleague in Japan gave us an antibody and We applied it both to our purified lutein binding protein preparations and also Applied it to monkey and human retinas and sure enough this antibody to a silkworm to a silkworm protein Specifically labeled now the inner segments right where we would expect of the inner segments of the photo receptors And even some labeling down into the Henley fiber layer here So this was pretty remarkable and gave us a lot of clues of what we should be looking at that Hopefully our protein might be a star D protein but The problem is there are many star D proteins in the human genome. They're exactly 15 So we had to sort out which of these 15 might be the important one because we weren't getting good sequence data on this And shown here is just from a review article just showing the different classifications that star D1 and star D3 were the ones that Are one family and you can see the other families here So we looked at we did computer modeling first and just did a Query using CBP the silkworm protein to see which of the 15 star D proteins in the human genome are Closely related to it and it came out that it showed that we had to focus primarily on star D1 and star D3 Which do show remarkable homology It's 30% homology between the silkworm protein and the human protein Which is really pretty good considering how separated the two the two organisms are and We then did West we obtained antibodies to all 15 of these star D proteins And that's shown here and look to see which ones were found in human tissue Which ones were found in mouse retinal tissue and also we did some positive controls with liver And we found that star D1 and star D3 were good candidates But star D1 was rapidly dropping out as one that we wanted to pursue. It's not found anywhere in anyone's retina It's not even found in the liver Actually star D1 is found primarily in adrenal gland and also very interestingly for some of our future work It's found in a different carotenoid rich tissue So what other tissue is anyone want to chime in is the most carotenoid rich in the besides the eye in humans Nope skins not not the most corpus luteum the yellow the corpus luteum of the ovary Is the most carotenoid rich and that's and that's where star D1 is and so we've established a collaboration now With the OBGYN department to look at the role of star D1 Perhaps in human fertility But star D3 was looking pretty good It's found very heavily expressed in the human macula less in the human peripheral retina And also in the RPE and the only other star D protein that's found in there is star D8 Now star D proteins are in bind small hydrophobic ligands So this is this was all looking pretty good that we needed to focus on this so We obtained and this is just showing the Western blot showing the ant and a different antibody using against star D3 That's more specific. You can see a strong labeling of star D3 right in the human macula Normalized human peripheral retina has almost has very little although it is detectable and very interestingly the mouse I told you they can't pick up carotenoids. They don't express star D3 in their retina here And again, we could confirm this also. It's a little bit out of line here that Star D3 is expressed by RTPCR So is it found in the retina and this is some beautiful work done by Gene Frederick here In our department where we labeled the cones just all cones in green to see what everything is is But we use the antibody to star D3 shown here in red and you can see that it labels very nicely the cone inner segments and Into the Henley fiber layer right in a monkey fovea here There's a little bit of non-specific staining of the RPE because you can see if we pre-absorb Using the star D3 protein to get it to we lose all the staining as you would expect in the inner segments where this protein is There's a little bit of autofluorescence of the pigment epithelium. So it's localized in the right area and Shown here is a little is a more classic study here where you can see the red Right in the foveal pit that matches up really quite well with the distribution So the question is does it does star D3 actually bind the carotenoids? Well, I told you we did work before you doing brute force biochemistry where we would take the protein Incubate it overnight extract all of these it was and it took a long time to do all of those binding studies But we've upgraded to the modern age now and we did now use a method called surface plasma and resonance And we have a device here although we've upgraded again and on this you put your protein If you as long as you have a pure protein you put it on a on a gold chip right here And you've then passed through a micro capillary your ligand the solubilized carotenoid and When it binds to the protein right on the chip you get a change in refractive index That's very sensitively measured here And so we can look at binding now instead of looking at equilibrium binding overnight We can look at the actual time course of binding and release of the compounds and shown here is a That's what's called a sensor gram using GSTP one on the chip where you can see as we increase the concentration of carotenoids We can see a progressive change saturated change of response in these protein and the when zeaxanthin binds When mesozeaxanthin binds, but then when you put lutein on there all you get is noise This protein is exquisitely sense exquisitely selective for for zeaxanthin and mesozeaxanthin and We obtained good quality star d3 from that we expressed in our in in our laboratory And we did the binding protein studies and this just summarizes the differences between binding Basically, you want very low the lower the number the better that shows specific binding and you want to see some sort of Specificity between distinguishing between carotenoids. So human serum albumin, which is a non-specific binder doesn't bind Doesn't have very high affinity binding You don't see anything below 0.5 micro molar and it doesn't select between any of the carotenoids here The silkworm binding protein on the other hand can select out lutein and has a pretty high affinity at 0.18 GSTp1 is exquisitely Selective for zeaxanthin and mesozeaxanthin, but doesn't care about these other carotenoids and star d3 itself Although not quite as good as CBP Does show that it is selective for lutein compared to all the other carotenoids at least at a reasonable level. So Basically, we've been able to show and we're just about we're hopefully going to publish this within the next month It's in the revision stage right now that star d3 fulfills the three essential criteria to be the macula's lutein Binding protein it's it's macula enhanced on Western blots It's immunohistochemistry puts it right into the right layer of right areas and it selectively binds lutein with high affinity So in summary we've shown that there's specific satchel xanthin binding proteins are present in the human macula They're in the right area GSTp1 is the zeaxanthin binding protein and Star d3 is the lutein binding protein and very interestingly We've been able to show a whole new class of proteins the star d proteins play a prominent and recurring role in as lutein binding proteins In a wide variety of organisms ranging from silkworms to humans And so next we're beginning to start to look at what is the role both genetically and physiologically in these In the of these proteins in AMD and risk progression and that's for future studies So to just end up with I'll talk briefly very briefly about herbal medicines and age-related macular degeneration Which is certainly frontier out there but there is the problem of course is that objective evidence is generally lacking and But our patients come in and talk about at least some of the more popular herbals like billberry red wine I bright and goji berries So what do we know about billberry? Well billberry has been promoted to enhance dark adaptation and treat age-related macular degeneration They're not carotenoids. They're at the cyan cyanide in flavonoids and shown here's an example here There's been anecdotal reports that the Royal Air Force pilots in World War two ate lots of billberry And that's why they could see so well at night They also they didn't happen to mention that they also had radar which helped them quite a bit at night, too And that was the billberry is thought actually to have been a cover-up So they didn't have to talk about the radar that they actually had There's still no good prospective studies yet that we can advise our patients that it's important that it's useful in AMD But it's certainly being studied It's not found in very high amounts in the retina Resveratrol shown here in red wine I mentioned that epidemiology shows that there's somewhat lower levels of AMD and red wine drinkers But this is some the doses that are being given are very high But people are looking at this in future in in epidemic in prospective studies, I think right now Eyebride is another one that patients may mention It's promoted for both surface and retina disease It's can be used in compresses or teas or tea There's no objective mechanism I can find except for the fact it's got a good name for marketing And then finally you do have to remember gochie berries Which are found in the which have been known I think for centuries in the Chinese traditional medicine have been long promoted for eye disorder And this also known as wolfberry is usually consumed as dried fruit or tea, but this one actually does make sense It's extraordinarily rich in Zezanthin in fact It's a commercial source of Zezanthin in some in some areas So this one you can see that traditional medicine did get it right that this one probably has could be important for eye disease So to sum up, what do we tell our patients now? What's the state of knowledge still when I talk with my patients? I tell them to eat a healthy diet with lots of fruits and vegetables and fish and no excessive fat I In patients who are at high risk of progression They need to consider an arid supplement plus I I'm not willing to wait for the results of arids to I feel comfortable recommending Lutein and Zezanthin at these doses the six to ten milligram lutein dose and two milligrams of Zezanthin Fish oil There's not as strong an evidence, but it's certainly reasonable to take I have to tell the patients to wait on single nutrient supplements until there's more data Alcohol may be important, but obviously should only be used in moderation Know a smoking is very bad and avoid excessive light exposure, and I certainly support support Thank our arids to participants where help will help give us definitive answers for our patients And I of course want to thank the people of my laboratory and my various collaborators and the Moran Eye Center. Thank you Question or two That's a good question. We do not know that yet We that's a little more difficult to measure the levels of the binding proteins at least on autopsy eyes And we can't do it non-invasively, but we're beginning to look at the expression of this. It's a whole It's a difficult problem. That's gonna be probably the Goal of my next grant to figure this out Yes, actually you were next You know mainly it has to do with their antioxidant effect as far as we know Degradation of carotenoids does happen, but it's relatively they're pretty stable, especially when they're bound to proteins So most of their antioxidant effect does not involve degradation It's a reversible It's a reversible cycle process and even the oxidized carotenoids that we find can probably be reduced back to their By some sort of antioxidant by some sort of cycle. So as far as we know in terms of inflammation and You know that it probably has to do with oxidative stress at least as far as we know Yeah, I didn't even talk about that that's a whole other whole other area So on the serum they're thought to be on HDL and LDL there to transport into the cell The thought is that scavenger receptor class the SRB one and CD 36 are being are somehow involved at least in Cell culture studies they seem to promote uptake of carotenoids See SRB one is not found in the rights is found in the pigment epithelium, but not in the retina in the right cells So it may be CD 36, but that's a whole other area. We're just barely getting involved in that area So I didn't want to talk about that right now But clearly we found just one part of we're solving parts of the puzzle. It's gonna take more to figure it all out Yes So with the the question is what about the blue light the yellow lenses that we put in it's It makes some Physiological sense that they may be important the the yellow pigmentation at least in the macula is acting as a screening compound There are a few other animals For example the if you look at squirrels, they don't have they have a very cone-rich retina They don't have carotenoids, but they have yellow lenses from chineurinic acid so physiologically it makes sense the thing that I'm disappointed in is that You know alcon and all the others went to marketing so quickly and never really did there's not been very good studies to show That they actually do make a difference. There's some studies that are coming out of Japan that are showing And more kind of intriguingly that patients who get when they compare clear lenses to yellow lenses Have a drop in their macular pigment level if they have the clear lenses So there may be something going on in the macula itself. That's long-term causing either degradation or low levels of macular pigment so my feeling is It's intriguing, but it's not proven, but they were able to get it through without without the proof All right. Well, thank you very much