Abstract
Cataracts are the leading cause of blindness worldwide. With an aging population, the incidence and prevalence of cataract-induced blindness is expected to rise considerably. L-carnosine (β-alanyl-L-histidine) is an endogenous dipeptide compound in vertebrates that has been designated an “antiaging” molecule due to its ability to delay senescence of cells. L-carnosine and N-acetyl-L-carnosine have demonstrated a reduction in opacification of the lens when used as a direct instillation to the eye. These compounds represent a promising new means of addressing cataracts.
Introduction
Cataract is the opacification of the ocular lens or capsule. According to the World Health Organization, in the year 2002, the last year that statistics were estimated, cataracts caused nearly half of the 37 million cases of blindness worldwide.1 In the United States, 20.5 million Americans over the age of 40 are affected in at least one eye, with an estimated increase to over 30 million Americans by the year 2020.2 Surgery is the only treatment option available and, while highly successful at restoring sight, it is not feasible in many developing countries that lack the infrastructure for adequate access to care.3 Ultimately, an easily disseminated medication that is economical and convenient could have a profound impact on the prevalence of the disease.4 Currently no agent is approved for use in preventing or delaying the onset of cataracts. There is evidence, however, that the natural agent L-carnosine (β-alanyl-L-histidine) effectively delays cataract development. The uniformity of benefit from preliminary evidence on carnosine and lens health, combined with a low toxicity profile make this endogenous dipeptide an intriguing candidate for prevention of cataract formation.
Background
Lens fiber cells are long, transparent cells continually produced by epithelial cells in the anterior portion of the lens and growing around the periphery (like longitudinal lines on a globe) to reach the posterior aspect. This process begins embryologically with the first lens fiber cells essentially providing the nexus upon which fetal and then adult lens fibers are built. Anatomically, these cells accumulate like layers in an onion, with the center becoming the nuclear region, outside of which is the cortical region. Aptly named, the subcapsular region is found between the cortical region and capsule that encompasses the lens.
Of note, all mature lens fiber cells lack nuclei or organelles, necessitating diffusion of many molecules, including nutrients, into the cells. The lack of intracellular structures and complete solubility of proteins, predominantly proteins called crystallins, within the lens cells is necessary for light rays to penetrate to the back of the eye.
Cataracts are classified into 3 types, depending on the region of the eye in which they originate.
Cataracts are classified into 3 types, depending on the region of the eye in which they originate. Nuclear cataracts begin at the center of the lens and affect distant vision in particular. These are the most common type of cataract and are thought to occur due to the aging process—thus the common term “age-related” or “senile” cataracts. Cortical cataracts are also called “diabetic cataracts”; they begin in the periphery of the lens and progress inward in a spoke-like fashion. Subcapsular cataracts usually begin at the back of the lens (posterior subcapsular cataracts) and are found in patients with a history of diabetes, steroid use, retinitis pigmentosa, or severe nearsightedness.
While symptomology varies between type and degree of cataract, the main symptoms of cloudy vision and sensitivity to glare is common to all forms at some stage of development. Double vision or multiple images in one eye can also occur. Unique to the development of nuclear cataracts, there may be a period of “second sight,” when the focus of near objects improves. This ability to see close objects more clearly is short lived, however, as the lens becomes more opaque and cloudy vision more severe. Cortical cataracts affect both distant and near vision and, while blurring occurs, sensitivity to glare and loss of contrast are more significant in this type of cataract. Subcapsular cataracts usually occur concomitantly with nuclear or cortical, and this type is marked by a more rapid progression.
Before any agents, synthetic or natural, are considered it is prudent to address known risk factors for cataract development. Risk factors include aging, diabetes, obesity, exposure to UVB radiation, smoking, previous eye surgery, prolonged use of some drugs (eg, corticosteroids, some diuretics, antipsychotics), and previous eye injury or inflammation. While many of these can be actively addressed through lifestyle modifications, others such as aging and eye injury clearly cannot be controlled. In addition to these established risk factors, epidemiological data suggests dietary intake of several nutrients can be protective. Nutrients such as taurine, carotenoiods, tocopherol, and acsorbate have shown a protective role from the development of some types of cataracts.5,6
Carnosine is an endogenous dipeptide synthesized by carnosine synthase in an ATP-dependent reaction that links amino acid substrates histidine and β-alanine. A high concentration is found in skeletal muscle fibers. As such, L-carnosine is also obtained from meat in the diet, although it appears to undergo rapid degradation by serum carnosinase. L-carnosine is also found in neural tissue, with a larger concentration in the olfactory bulb. Its biological role is not known conclusively, but the higher concentrations in these long-living cells is in keeping with its putative role as an antisenescence molecule.7
The presence of endogenous carnosine in the lens of the eye suggests it is needed in normal physiological processes.8 Of note, there is no evidence that the eye contains carnosine synthase. Therefore carnosine, like many other nutrients, must be derived from systemic circulation, diffusing into the aqueous humor then subsequently into the lens cells themselves. Carnosinase, the enzyme that degrades carnosine into its component amino acids, is found in the eye. The presence of carnosinase may result in rapid degradation of carnosine into its component amino acids.
L- Carnosine versus N-acetylcarnosine
Currently, N-acetylcarnosine (NAC), not L-carnosine, eye drops are commercially available. NAC was found to be a prodrug of L-carnosine within the eye, as it undergoes deacetylation to release carnosine into the aqueous humor.9 Since carnosine is thought to be rapidly degraded by resident carnosinase in the lens, and NAC appears to be protected from this enzyme, the instillation of NAC was proposed as a means of achieving a more effective delivery of L-carnosine to the lens. Furthermore, NAC is a less hydrophobic molecule, so it may more readily pass through the membrane lipid bilayer of lens cells. While the data on these compounds is certainly not interchangeable, when considering the compounds as an instillation directly into the eye, the molecular effects of L-carnosine can be extrapolated to include that seen with NAC as well, since the latter compound is completely deacetylated in situ to render L-carnosine.
Overview of Cataractogenesis and Carnosine
The development of cataracts, or cataractogenesis, is marked by many of the same processes that are involved in the senescence of cells. Namely, reactive oxygen species (ROS), advanced glycation end products (AGEs), and deleterious aldehydes and thiols accumulate intracellularly. In excess, these incendiary molecules lead to changes in lipid and protein structures that are the hallmark of aging cells. Two morphological changes that directly result in increasing opacification are peroxidation of the lipid bilayer of cellular membranes of the lens and crosslinking of otherwise soluble crystallin proteins forming insoluble aggregates within the lens fiber cells. Each of these macromolecular changes leads to disruption in the passage of light through the lens by scattering light rays, and with adequate accumulation, this eventually can obscure light passage completely.
The ability of L-carnosine to attenuate the products of lipid peroxidation within the ocular lens was first suggested by Dr. Alan Babizhayev and colleagues in 1987.10 Indeed, much research has since confirmed that carnosine reduces the formation of lipid peroxides within the lens.11,12,13
In addition to directly attenuating membrane structure damage through lessening peroxidation of lipids, reduction of lipid peroxides also reduces the formation of its highly reactive metabolite, malondialdehyde (MDA). MDA interacts with amino acid moieties on crystallin proteins within the lens to cause crosslinking of these proteins, resulting in insoluble aggregate molecules.14
In cataracts, many enzymes that normally provide defense against ROS overproduction, including superoxide dismutase and catalase, are depleted.15 Exacerbating this effect, the nuclear region of the lens is normally dependent on the production and subsequent diffusion of glutathione from the cortical region, but this diffusion process becomes less efficient as we age.16 Carnosine has been shown to preserve levels of these enzymes in cataract lenses, thus improving their antioxidative capacity.17 There is also postulation that carnosine lessens ROS damage through its ability to chelate free metal ions, which are required to generate O2-.18
Separately, carnosine has been shown to act as an alternative target for glycation, a “sacrificial transglycation,” that effectively binds sugars to itself rendering them unavailable to bind proteins. This leads to a measurable decrease in AGEs and may be responsible for much of carnosine’s antisenescence action apart from its antioxidant capacity.19
Another mechanism by which carnosine is able to reduce protein adducts is by directly binding to carbonyl groups on proteins. Carbonyl groups increase in high oxidation and glycation environments; thus they often coincide with aging of cells. Carnosine’s binding to reactive carbonyls, a process called “carnosinylation,” prevents proteins from binding to one another and forming adducts.20
Remarkably, L-carnosine has been shown capable of deaggregating the crystalline lens proteins that directly result in opacity. Methylglyoxal-induced glycation of α-crystallin aggregates was reversed with the addition of carnosine to the media.21 The ability of carnosine to denatured protein aggregates was also demonstrated in an in vitro study of rat lenses. Both L- and D-carnosine had a “disassembling” effect on α-crystallin fibrils and restored the transparency of cataractous lenses. This was accompanied by a reduction in the average size of the proteins, confirming that disassembly did take place.22
A dominant pathway of cataractogenesis in diabetic patients, in addition to those discussed above, is the polyol pathway. This is the sequential transformation of glucose into sorbitol and subsequently fructose within the lens by the enzymes aldose reductase and sorbitol dehydrogenase, respectively.23 Aldose reductase inhibitors have been found to prevent sugar-induced cataracts, thus confirming the integral role of this pathway on cataract formation.24 The fructose molecule is highly reactive and, together with oxidized proteins and a direct reduction of esterase activity, leads to AGEs. Carnosine has been shown to directly inhibit aldehyde reductase, thus lowering the concentration of fructose and AGEs.25 Further, the addition of carnosine to lens media abrogated fructose induced deactivation of esterase activity.26
In Vivo Studies
In one study using rats with streptozocin-induced diabetes, cataract development was shown to progress in a biphasic manner, with a slow progression in the first 8 weeks followed by a rapid increase in the next 5 weeks. Carnosine delayed the onset of lens opacification in the early stages of cataract development, reaching statistical significance at week 4 (P<0.05), but failed to affect later progression of the disease. This rodent study also demonstrated a reduction in the levels of AGEs in treated eyes versus untreated, which was concordant with better preservation of glutathione and catalase levels in the treated groups.27
NAC may not only act as a progdrug but may itself be involved in the antiaggregation effects observed with NAC-containing eye drops. A rodent study using UV-induced cataractogenesis supported this finding and suggested that a mixture of D-pantethine and NAC is even more effective than NAC alone in its antiaggregate effects.28
A recent study using rats with streptozocin-induced diabetes showed a delay in the development of cataracts using an instillation of aspirin (1%), L-carnosine (1%), or an alternating combination of the 2. Both single agent interventions delayed cataracts while the combination was more effective than either agent alone. In addition, there was an increase in the levels of soluble protein in the lenses of the treated groups versus controls.29
In a small study, 30 dogs of various breeds with existing lens opacities were administered a combination product containing 2% NAC. Additional ingredients in the proprietary formula include glutathione, cysteine ascorbate, L-taurine, and riboflavin (Ocluvet™, by Practivet, Arizona, USA). 58 eyes of 30 dogs were evaluated, 22 with mature cataract, 13 with immature cataract, 9 with cataract associated with eye inflammation, and 14 with nuclear sclerosis. Images of the lenses were taken at weeks 2, 4, and 8. Objective measurement of lens opacification was determined by a lens opacification index (LOI) using computerized integration of the grayscale level of each pixel across the lens image. There was a reduction in the level of LOI in all groups, although this was only statistically significant in the immature cataract and nuclear sclerosis groups. Subjectively, owners perceived improvement in 80% of the dogs studied. The greater benefit in early cataract development supports in vitro data that suggests later stages of cataract development appear to overwhelm the benefits of antioxidant support.30
Another canine study using 1.0% NAC in its patented formulation (Can-C®, Innovative Vision Products, Delaware, USA) used 30 dogs in the treatment group, 15 dogs in placebo-controlled group, and 10 dogs without treatment. All dogs had cataracts at the start and all eyes were assessed. Placebo consisted of all ingredients in the drops except NAC. After 6 months of treatment, 96% of eyes in the treatment group showed improvement in the slit image and retroillumination photographs. No mention is given in the publication of the statistical analysis or of the results of the 2 nontreatment groups.31
Babizhayev and colleagues have shown beneficial effects of 1.0% NAC instillation in the eyes in several small human clinical trials. In a randomized study of 49 subjects with senile cataracts (76 affected eyes), 26 (41 affected eyes) were given NAC (1.0%) eyedrops twice daily. There were 2 control groups: a placebo group of 13 patients (21 affected eyes) who received an eye drop formulation containing all ingredients but NAC and an untreated group of 10 patients (14 eyes) who did not receive any eye drops. For statistical analysis, the control groups were pooled. The 6-month outcome showed 90% of patients in the treated group had improved visual acuity and 89% showed improved glare sensitivity. Image analysis of the cataracts at 6 months showed a statistically significant difference (P<.0001) using an in-house imaging that included slit-lamp assessment and retroillumination of the lenes to assess.32
In a continuation of the above study, the participants were assessed every 6 months for a total of 24 months. At 24 months no one in the treatment group had declining visual acuity, while overall there was a decline in the control group. Overall, reductions in glare sensitivity and visual acuity were sustained for the 24 month duration of the study; this difference was statistically significant (P< .0001).33
In another trial by the same group, 65 older drivers with 1 or both eyes affected by cataract and 72 older adult controls without cataract were recruited to participate in a double-blind, placebo-controlled trial. Assessment of glare sensitivity (halos) at red and green targets was made using an in-house test. At 4 months participants receiving NAC had a statistically significant improvement in visual acuity and glare sensitivity (P<0.001).34
In 2009 another trial of 75 patients with cataracts and 72 without, 1.0% NAC was instilled daily for 9 months. In both groups visual acuity and reduction in glare sensitivity reached statistical significance (P<0.001) at 9 months.35
Discussion
A growing body of evidence suggests that L-carnosine attenuates aging processes, ultimately culminating in delayed senescence of the organism as a whole, as suggested in Drosophila and mouse models.36 Mechanisms of carnosine’s antiaging effects include its role as an antioxidant, antiglycating agent, metal chelating agent, aldehyde scavenger, carbonyl scavenger, and stimulator of nitric oxide synthase.37 Many of these are relevant to the development of cataracts, and it is a reasonable assumption that carnosine’s beneficial effects in the lens are largely due to these mechanisms of cellular self-preservation. For a thorough review of carnosine’s biological effects throughout the body, the reader is referred to a recent publication by Alan Hipkiss.38
Much of the clinical research demonstrating the efficacy of carnosine in preventing or treating cataracts in humans has been done by Dr. Babizhayev of Innovative Vision Products (IVP), Delaware, USA. His group proposed the use of NAC as a prodrug for L-carnosine in 1996.39 Since then, IVP has manufactured a patented formula called Can-C (private label, Nu-Eyes) which consists of deionized water, glycerin (1.0%), NAC (1.0%), carboxymethylcellulose (0.3%), benzyl alcohol (0.3%), and potassium borate and bicarbonate buffers.40 Babizhayev’s group proposes this particular formulation obtains superior absorption through the cornea and that acetylation of carnosine optimizes the delivery of carnosine into the lens in several ways: protection from degradation by carnosinase in the aqueous humor, a more lipophilic molecule that allows for easier penetration into the lens, and, due to the pharmacokinetics of deacetylation, “timed release” of the carnosine molecule.41,42
Babizhayev and colleagues reported that instillation of non-acetylated L-carnosine (1%) to rabbit eyes did not lead to an increase of L-carnosine concentration in the anterior compartment versus placebo, an effect they speculate is due to carnosinase degradation of the dipeptide.43 While this is in keeping with the assumption that carnosinases in the eye rapidly degrade L-carnosine, there is recent evidence of L-carnosine accumulation in the eyes of rabbits using a 5.0% instillation.44 Further, Babisheyev asserts that not only is L-carnosine less effective than NAC, but that there may be risk of harm to the eye due to the byproduct of histamine that ultimately results from the degradation of carnosine. These predictions are disputed, at least in part, by data in animals that demonstrates reduced cataractogenesis with the use of L-carnosine eye drops.45 There is no in vivo data that directly compares the efficacy of L-carnosine versus NAC eyedrops. More definite studies are needed to unequivicably conclude whether NAC is superior to L-carnosine.
While there is no data on dietary consumption of carnosine and cataract development, serum carnosine levels do decrease with age.46 Given carnosine’s multiple antisenescent actions, this correlation may be part of the aging process itself. Carnosine levels can be increased through the consumption of meat, and a carnivorous diet has been postulated to have possible antiaging effects.47 Strict vegetarian diets are devoid of the peptide, so a diet high in carnosine precursors histidine and alanine, with emphasis on the more rate-limiting alanine, may be the closest simulation. Oral supplementation of carnosine or its substrates has not been studied in regard to eye health. There is also no data to suggest a diet high in carnosine will effectively diminish cataract formation.
Conclusion
The endogenous dipeptide carnosine (β-alanyl-L-histidine) is recognized as an antiaging molecule at both cellular and whole animal levels. Overall, the state of the evidence on carnosine’s role in delaying or treating cataractogenesis is preliminary. However, the role of carnosine as a universal antiaging molecule is fairly well established. This larger body of evidence, along with the uniformity of beneficial effects on cataractous lenses in the limited studies that have been published, provide rationale for consideration of this nontoxic agent in delay of cataract development, particularly in early stage cataracts.
