Various human diseases and conditions have ocular components. The human eye lens is a transparent, biconvex structure that helps to refract light to be focused on the retinal surface. The change in curvature helps in adjusting the focal distance of the eye so that it can focus on objects at different distances. This adjustment of the lens is called accommodation. Presbyopia is a common ocular condition observed in patients above 50 years and is characterized by loss of flexibility of the crystalline eye lens and in turn, accommodation. Cataract is the leading cause of blindness, affecting 40 million people worldwide. It is a multifactorial ocular disease caused by genetics, age, and environment. There are reports that glycation gradually damages the lens by causing aggregation of the lens proteins.
Diabetes Mellitus
Diabetes mellitus is an endocrine metabolic disorder characterized by high blood sugar levels which give rise to complications in the eye, kidneys and the brain. Diabetes triggers the development of ocular diseases, for example, diabetic retinopathy, glaucoma and cataracts which are the leading cause of blindness around the world. The most common method for the diagnosis of diabetes involves measuring the blood sugar levels in the body. One major disadvantage of this method is that blood sugar levels fluctuate which contributes to false negative results. This leads to delay in treatment, eventually causing permanent damage to the organs. Therefore, diagnosis of diabetes at an early stage is very crucial. Additional or alternative diagnostic tests would be beneficial.
Diabetes arises due to inadequate insulin production, or because the body's cells are non-responsive to insulin, or both. Globally, about 382 million people have diabetes, of which around 46% remain undiagnosed. Diabetes mellitus starts a vicious cycle of diseases affecting the heart, kidneys, eyes and the nervous system. The high blood sugar levels have been observed to cause damage to small blood vessels in these organs by destroying their structure. The total estimated cost of diagnosed diabetes in 2012 is $245 billion which increased to $548 billion in 2013. Overall, the number of diabetics as well as the expenditure including direct and indirect costs have been increasing drastically.
Glycated Proteins
In addition to blood sugar levels, there are several other methods used for the diagnosis of diabetes. Measurement of glycated proteins, primarily glycated hemoglobin (HbA1c or A1C), has been widely used for routine long-term monitoring of glucose control and as a measure of risk for the development of diabetes complications. The A1C test measures average blood glucose for the past 2 to 3 months and, if the values are greater than or equal to 6.5%, the person is considered diabetic. The Fasting Plasma Glucose (FPG) and Oral Glucose Tolerance (OGT) tests measure the blood sugar levels after fasting and having a sweet drink respectively. Values greater than or equal to 126 mg/dl and 200 mg/dl are considered diabetic in FPG and OGT respectively. (Table 7)
Although, these tests give accurate results in diabetic patients, they have their exceptions. The blood glucose levels in the body fluctuate depending on the meals, exercise, sickness, and stress. It has also been shown that different diagnostic tests might give varying results and not agree with one another. The glycation of hemoglobin occurs at several amino acid residues and, as a result, several adducts of hemoglobin A (HbA) and various sugars are formed by the non-enzymatic post-translational glycation process. This process involves the formation of a labile Schiff base intermediate followed by the Amadori rearrangement. The reaction is slow, irreparable, and the reaction rate depends on the ambient glucose concentration. Also, these tests do not take into consideration that the proteins, especially in the vasculature, have rapid turnover and hence are not always the same over time.
HbA1c has been recommended as an accurate and precise marker for diabetes based on advances in instrumentation and standardization. The theory behind the A1C test is that red blood cells live an average of three months. So, if the amount of glycated hemoglobin is measured, results will give an idea of glycation that occurred over the last 3 months. But, research has shown that the lifetime of red blood cells of diabetics is comparatively shorter than that of non-diabetics. This means that the hemoglobin turnover is faster in case of diabetics, and therefore is a major disadvantage for diagnosing the patients in their early stages. Also, it has been reported that people with hemoglobin variants, for example, HbC and HbS have shown false negatives. A false elevated A1C level has been observed in certain clinical situations that affect RBC life span with iron deficiency anemia, high alcohol consumption and hypertriglyceridemia. Other cases which have shown false results include patients with kidney failure and liver disease.
Ocular Proteins
Three major proteins called α-, β- and γ-crystallins are found in the eye lens. The structure, biochemical and physiological properties as well as functionalities of these crystallins have been reported. The monomeric γ-crystallins are globular and the smallest with a molecular weight of about 20 kDa. In the case of β-crystallins, their subunits form oligomers with low molecular weight species (βL-60 kDa) and high molecular weight species (βH-160 kDa). α-crystallin is the most abundant and largest of the lens proteins (˜18 nm in diameter) consisting anywhere between 30-40 subunits with molecular weight ranging from 800-1200 kDa.
Due to a very low protein turnover, crystallins are considered to be some of the longest-lived proteins in the human body. Because of the long half-life, α-crystallin is prone to irreversible modifications leading to changes in structure and function. The most commonly observed post-translational modifications include photo-oxidization, deamidation, racemization, phosphorylation, acetylation, glycation and age-dependent truncation. Post-translational modifications alter protein-protein interactions and subsequently destabilize and reduce the solubility of native crystallins.
Alpha-Crystallins
The eye lens is avascular and constitutes a dense matrix of closely packed proteins. α-crystallin is a major water soluble small heat shock protein (˜45%) found in the eye lens. It is isolated from vertebrate eye lens as a polydisperse, hetero-oligomeric complex of approximately 800-1200 kDa, consisting of 35-40 subunits. It is made up of two distinct sub-units—A and B in the ratio of 3:1 respectively. It has a chaperone function, protecting other proteins and crystallins from thermal aggregation. This in turn helps in maintaining the transparency of the eye lens. Recently, it has been observed that α-crystallin sub-units are not restricted to the eye lens, but also are expressed in other non-lenticular tissues like retina, heart, brain and kidneys. While αA is mostly restricted to the lens and retina, it has been reported that αB subunit is expressed ubiquitously in cells undergoing stress.
In its native form, α-crystallin consists of two homologous subunits showing 55% sequence similarity in a ratio of 3:1—αA and αB, with 173 and 175 amino acid residues, respectively. The molecular weight of these two subunits is approximately 20 kDa. αA crystallin is confined to the lens with a small amount in the retina, spleen and thymus. αB crystallin is ubiquitously present in the lens, retina and the heart abundantly, and is expressed under stress and pathological conditions in the spinal cord, muscles, brain and the kidneys. There are reports that α-crystallins act as anti-apototic regulators and prevent apoptosis under stress conditions, thereby protecting the tissues from damage
A recent study has detailed how in concentrated suspensions of alpha crystallin, inter-particle correlations are well described by the structure factor for a hard sphere fluid.
The chaperone function of α-crystallin has been reported to prevent thermal aggregation of other proteins. Over a period of time, α-crystallin undergoes irreversible post-translational modifications of which non-enzymatic glycation is prominent especially in aging and diabetes. As a result, the protein slowly starts losing its chaperone ability and starts to aggregate. Glycation also leads to loss of anti-apoptotic activity of alpha crystallin.
Although α-crystallin has been studied extensively, the quaternary structure of the native protein has not been elucidated. As a result, the location of protein modifications which are a part of disease pathology are not resolved. As the lens is avascular and has no turnover, the modifications that occur in the lens alpha crystallin due to non-enzymatic glycation are permanent. Reducing sugars react with basic amino acids of proteins to form Schiff's bases which undergo rearrangement to Amadori products and finally form advanced glycation end-products (AGEs). These AGEs lead to loss of protein integrity, increase hydrophobicity and play an important role in protein denaturation.
Protein denaturation is usually associated with the formation of aggregates. Protein precipitation and aggregation involves the growth of large sized particles and hence is an optimum method for biophysical characterization based on particle size. Light scattering characteristics of protein aggregation of crystallin glycation effects on the protein and its role in the decrease of lens flexibility (presbyopia) as well as the formation of cataracts need to be determined.
An important biomarker for diabetes related diseases is the formation of advanced glycation endproducts (AGEs). AGEs are formed due to non-enzymatic glycation of the proteins on exposure to open chain sugars and dicarbonyl intermediates. Hyperglycemic conditions, oxidative and thermal stress lead to the formation of Schiff s bases with basic amino acids like lysine and arginine. Further, Amadori products are formed due to rearrangement of the Schiff's bases when highly reactive carbonyl intermediates accumulate and attack the amino and guanidine groups on the proteins. Unlike hemoglobin and albumin, the heat shock proteins have long half-lives and very low turnover. As a result, the accumulation of glycation products over a long period of time is likely of quantitative significance.
Methylglyoxal (MGO) is a glycating agent that is generated non-enzymatically from the oxidation and spontaneous dismutation of intermediates in the glycolysis pathway or enzymatic oxidation reaction catalyzed by peroxidases. MGO is reported to be toxic and to interfere with cellular mechanisms. It has been reported to impair functions of mitochondria and also produce reactive oxygen species. Another source of this dicarbonyl reactive intermediate in the body is deficiency of triose phosphate isomerase leading to elevated dihydroxyacetonephosphate (DHAP) levels observed in congenital hemolytic anemia and other neurodegenerative diseases. DHAP spontaneously disintegrates to methylglyoxal which acts as a strong agent in the formation of advanced glycation end products (AGEs). Because MGO reacts rapidly with the proteins, modification by MGO is a good in vitro model for investigating the long term effects of glycation on heat shock proteins. (FIG. 17)
USPTO Backgrounds
USPTO Backgrounds