WO2010132459A2

WO2010132459A2 - Biomarkers for assessment of age-related macular degeneration

Info

Publication number: WO2010132459A2
Application number: PCT/US2010/034398
Authority: WO
Inventors: John W. Crabb; Ram H. Nagaraj; Jiayin Gu; Jiaqian Ni; Xianglin Yuan
Original assignee: Cleveland Clinic Foundation; Case Western Reserve University
Priority date: 2009-05-11
Filing date: 2010-05-11
Publication date: 2010-11-18
Also published as: WO2010132459A3

Abstract

Biomarkers that can be used to assess the risk that a subject will develop age-related macular degeneration (AMD) and/or to assess the severity of AMD in the subject are described. The biomarkers are also useful in clinical trials to monitor the effect of a treatment and in pre-clinical trials to screen candidate therapies. The biomarkers can include carboxyethylpyrrole (CEP) adducts, carboxymethyllysine (CML), and pentosidine and be measured individually and in various combinations.

Description

BIOMARKERS FOR ASSESSMENT OF AGE-RELATED MACULAR

DEGENERATION

BACKGROUND

Age-related macular degeneration (AMD) is a progressive, multifactorial disease and the leading cause of severe vision loss in the elderly in industrialized countries (1). Deposition of debris (drusen) in the macular region of Bruch's membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE), is an early, hallmark risk factor. The disease can progress to advanced dry AMD (geographic atrophy), which is characterized by regional degeneration of photoreceptor and retinal pigment epithelial (RPE) cells, or to advanced wet AMD (choroidal neovascularization, or CNV), which is characterized by abnormal blood vessels growing from the choriocapillaris through Bruch's membrane beneath the retina. CNV accounts for over 80% of debilitating visual loss in AMD, however only 10-15% of AMD cases progress to CNV.

There is growing consensus that AMD is an age-related inflammatory disease involving dysregulation of the complement system, however, triggers of the inflammatory response have yet to be well defined. Oxidative stress appears to be involved as smoking significantly increases the risk of AMD (2), antioxidant vitamins can selectively slow AMD progression (3), and a host of oxidative protein and DNA modifications have been detected at elevated levels in AMD Bruch's membrane, drusen, retina, RPE and plasma (4-11). Oxidative protein modifications like carboxyethylpyrrole (CEP) and Λ^-carboxymethyllysine (CML), both elevated in AMD Bruch's membrane, stimulate neovascularization in vivo (12, 13), suggesting possible roles in CNV. Other studies have shown mice immunized with CEP protein modifications develop an AMD- like phenotype (14). Accordingly, oxidative modifications may be catalysts or triggers of AMD pathology (6).

Mounting evidence implicates a role for advanced glycation endproducts (AGEs) in AMD pathology (4, 5, 7, 15, 16). AGEs are a heterogeneous group of mostly oxidative modifications resulting from the nonenzymatic Maillard glycosylation reaction that appear to contribute to age- related diseases and diabetic complications (17, 18). In 1998, CML was the first AGE to be associated with AMD Bruch's membrane and drusen (4). Other AGEs have since been detected in AMD ocular tissues by multiple investigators (5, 7, 15), and in Bruch's membrane, drusen, RPE, and choridal extracellular matrix from healthy eyes (6, 19). CML and pentosidine, a fluorescent crosslinking AGE, increase with age in Bruch's membrane (15, 20). Receptors for AGEs (RAGE and AGE-Rl) also appear elevated on RPE and photoreceptor cells in early and advanced dry AMD (7), especially in RPE overlying drusen- like deposits on Bruch's membrane (16). RAGE is a pattern-recognition receptor related to other receptors known to impact AMD pathology, namely complement (21-27). AGE-R3 is elevated in advanced dry AMD Bruch's membrane (46).

While AMD susceptibility genes now account for over 50% of AMD cases (30), many individuals with AMD risk-genotypes may never progress to advanced disease with severe visual loss. Nevertheless, the prevalence of advanced AMD in the USA is projected to increase to ~3 million by the year 2020 (31). It would be highly desirable to have simple clinical tests for assessing the risk of progression to advanced AMD.

SUMMARY

Described herein are biomarkers that can be used to assess the risk that a subject will develop age-related macular degeneration (AMD) and/ or to assess the severity of AMD in the subject. The biomarkers are also useful in clinical trials to monitor the effect of a treatment and in preclinical trials to screen candidate therapies. The biomarkers can include carboxyethylpyrrole (CEP) adducts, carboxymethyllysine (CML), and pentosidine and be measured individually and in various combinations. In many cases in which CEP is measured, one can also measure autoantibodies directed against CEP.

Described herein is a method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level of N(6)-carboxymethyllysine (CML) in a bodily fluid of the subject;

(b) comparing the level of CML measured to a control value; and (c) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the control value or characterizing the subject as at lesser risk of developing AMD if the level of CML measured is not greater than the control value.

(a) measuring the level of pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a control value;

(c) characterizing the subject as at greater risk of developing AMD if the level of pentosidine measured is greater than the control value or characterizing the subject as at lesser risk of developing AMD if the level of pentosidine measured is not greater than the control value.

(a) measuring the level of pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) measuring the level of CML in a bodily fluid of the subject;

(d) comparing the level of CML measured to a CML control value; and

(e) characterizing the subject as at greater risk of developing AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CML measured is greater than the CML control value or characterizing the subject as at lesser risk of developing AMD if the level of pentosidine measured is not greater than the pentosidine control value and the level of CML measured is not greater than the CML control value.

Described herein is a method for assessing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the of level of N(6)-carboxymethyllysine (CML) in a bodily fluid of the subject;

(b) comparing the level of CML measured to a CML control value; (c) measuring the level of 2-(ω-carboxyethyl)pyrrole (CEP) adducts or anti-CEP antibodies in a bodily fluid of the subject;

(d) comparing the level of CEP adducts or anti-CEP antibodies measured to a CEP control value; and

(f) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the CML control value and the level of CEP adducts or anti-CEP antibodies measured is greater than the CEP control value or characterizing the subject as at lesser risk of developing AMD if the level of CML measured is not greater than the CML control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.

(a) measuring the level of pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) measuring the level of 2-(ω-carboxyethyl)pyrrole (CEP) adducts or anti-CEP antibodies in a bodily fluid of the subject;

(f) characterizing the subject as at greater risk of developing AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adducts or anti-CEP antibodies measured is greater than the CEP control value or characterizing the subject as at lesser risk of developing AMD if the level of pentosidine measured is not greater than the pentosidine control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.

(c) comparing the level of CML measured to a CML control value; (d) measuring the level of 2-(ω-carboxyethyl)pyrrole (CEP) adducts or anti-CEP antibodies in a bodily fluid of the subject;

(e) comparing the level of CEP adducts or anti-CEP antibodies measured to a CEP control value;

(f) measuring the level of pentosidine in a bodily fluid of the subject;

(g) comparing the level of pentosidine measured to a pentosidine control value; and (h) characterizing the subject as at greater risk of developing AMD if the level of

CML measured is greater than the CML control value, the level of CEP adducts or anti-CEP antibodies measured is greater than the CEP control value, and the level of pentosidine measured is greater than the pentosidine control value or characterizing the subject as at lesser risk of developing AMD if the level of CML measured is not greater than the CML control value, the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value, and the level of pentosidine measured is not greater than the pentosidine control value.

In various cases, the step of measuring CML in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid. In various cases, the step of measuring pentosidine in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid. In various cases, the step of measuring CEP adducts or anti-CEP antibodies in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.

In various cases the bodily fluid is selected from serum and plasma. In various cases, the subject is not suffering from diabetes.

In various cases, the method includes characterizing the subject as at lesser risk of developing AMD if the level of CML measured is greater than the CML control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.

In various cases, the method includes characterizing the subject as at lesser risk of developing AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value. In various cases, the method includes characterizing the subject as at lesser risk of developing AMD if the level of CML measured is greater than the CML control value, the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value, and the level of pentosidine measured is greater than the pentosidine control value.

In various cases, the AMD is advanced AMD and the control value is a control value for mild to moderate AMD. In various cases, the AMD is mild to moderate AMD and the control value is a control value for non-AMD. In various cases, the AMD is dry AMD. In various cases, the AMD is wet AMD.

The method can further include determining whether the subject carries a genetic marker associated with increased risk for developing AMD wherein the genetic marker is present in a gene selected from the group consisting of complement factor; complement C3; ARMS2; and MTND2.

In various cases, the characterizing step comprises characterizing the subject as having a less than 60% chance of developing AMD. In various cases, the characterizing step comprises characterizing the subject as having a greater than 60% chance of developing AMD.

Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the of level N(6)-carboxymethyllysine (CML) in a bodily fluid of a subject at a first time before administering a test compound and at a second time after administering the test compound;

(b) comparing the increase in the level of CML measured between the first and second time to a CML control value increase; and

(d) identifying the test compound as a candidate compound for treating or reducing the risk of developing AMD if the increase in the level of CML measured is less than the CML control value increase. Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level of pentosidine in a bodily fluid of a subject at a first time before administering a test compound and at a second time after administering the test compound;

(b) comparing the increase in the level of pentosidine measured between the first and second time to a pentosidine control value increase; and

(d) identifying the test compound as a candidate compound for treating or reducing the risk of developing AMD if the increase in the level of pentosidine measured is less than the pentosidine control value increase.

(a) measuring the level of N(6)-carboxymethyllysine (CML) in a bodily fluid of the subject at a first time before administering a test compound and at a second time after administering the test compound;

(b) comparing the increase in the level of CML measured between the first and second time to a CML control value increase;

(c) measuring the level of CEP adducts or anti-CEP antibodies in the serum of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(d) comparing the increase in the level of CEP adducts or anti-CEP antibodies measured between the first and second time to a CEP control value increase; and

(e) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.

In some cases step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.

(a) measuring the of level N(6)-carboxymethyllysine (CML) in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound;

(c) measuring the level pentosidine in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(d) comparing the increase in the level of pentosidine measured between the first and second time to a pentosidine control value increase; and

(e) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.

In some cases, step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.

(a) measuring the level pentosidine in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound; (b) comparing the increase in the level of pentosidine measured between the first and second time to a pentosidine control value increase;

(c) measuring the level of CEP adducts or anti-CEP antibodies in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(f) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of pentosidine measured is less than the pentosidine control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.

In some cases step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of pentosidine measured is less than the pentodine control value increase and (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase.

(a) measuring the level of N(6)-carboxymethyllysine (CML) in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound;

(c) measuring the level CEP adducts or anti-CEP antibody in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(d) comparing the increase in the level of CEP or anti-CEP antibodies measured between the first and second time to a CEP control value increase; and (e) measuring the level of pentosidine in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(f) comparing the increase in the level of pentoside measured between the first and second time to a pentoside control value increase; and

(g) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase

In some cases, step (g) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if two or more of: (i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.

In some cases step (g) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if :(i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adducts or anti-CEP antibodies measured is less than the CEP control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.

(a) measuring the level of N(6)-carboxymethyllysine (CML) in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound; and (b) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

(a) measuring the level of pentosidine in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound;

(b) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound.

(a) measuring the level of N(6)-carboxymethyllysine (CML) in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(b) measuring the level of CEP adducts or anti-CEP antibodies in the serum of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(c) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compoundand (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound. In some case step (c) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both: (i) the level of CEP or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

(b) measuring the level of pentosidine in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(c) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

In some cases, step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

Described herein is a method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising: (a) measuring the level of pentosidine in a bodily of a subject at a first time before administering the test compound and at a second time after administering the test compound;

(b) measuring the level of CEP adducts or anti-CEP antibodies in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(c) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

In some cases step (c) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound; and (ii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

(c) measuring the level pentosidine in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound; (d) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

In some cases, step (d) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if two or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound

In some cases step (d) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adducts or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

In some cases the step of measuring comprises the use of mass spectrometry; the bodily fluid is obtained from a biological sample obtained from the subject the subject is a human taking part in a clinical trial; the subject is a non-human mammal. Described herein is method for diagnosing age-related macular degeneration (AMD), the method comprising:

(b) comparing the level of CML measured to a control value; and

(c) diagnosing the subject suffering from AMD if the level of CML measured is greater than the control value.

(a) measuring the level of pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a control value;

(c) diagnosing the subject suffering from AMD if the level of pentosidine measured is greater than the control value.

(a) measuring the level of pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) measuring the level of CML in a bodily fluid of the subject;

(d) comparing the level of CML measured to a CML control value; and

(e) diagnosing the subject suffering from AMD if the level of pentosidine measured is greater than the control value and the level of CML is greater than the CML control value

(b) comparing the level of CML measured to a CML control value;

(c) measuring the level of 2-(ω-carboxyethyl)pyrrole (CEP) adducts or anti-CEP antibodies in a bodily fluid of the subject; (d) comparing the level of CEP adducts or anti-CEP antibodies measured to a CEP control value; and

(f) diagnosing the subject suffering from AMD if the level of CML measured is greater than the CML control value and the level of CEP adducts or anti-CEP antibodies is greater than the CEP control value

(a) measuring the level of pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) comparing the level of CML measured to a CML control value;

(d) measuring the level of 2-(ω-carboxyethyl)pyrrole (CEP) adducts or anti-CEP antibodies in a bodily fluid of the subject;

(f) measuring the level of pentosidine in a bodily fluid of the subject;

(g) comparing the level of pentosidine measured to a pentosidine control value; and (h) diagnosing the subject suffering from AMD if the level of CML measured is greater than the CML control value, the level of pentosidine is greater than the pentosidine control value, and the level of CEP adducts or anti-CEP antibodies is greater than the CEP control value

In various cases, the patient is diagnosed as not suffering from AMD if the level of CML measured is greater than the CML control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.

In various cases, the patient is diagnosed as not suffering from AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value.

In various, case the patient is diagnosed as not suffering from AMD if the level of CML measured is greater than the CML control value, the level of CEP adducts or anti-CEP antibodies measured is not greater than the CEP control value, and the level of pentosidine measured is greater than the pentosidine control value.

DESCRIPTION OF THE DRAWINGS

Figure 1. CML and Pentosidine Are Elevated in AMD Plasma. CML (A) and pentosidine concentrations (B) quantified by LC-MS/MS and LC-fluorimetry from control (n = 30) and AMD (n = 60) plasma donors and autoantibody titers for CML (C) and pentosidine (D) quantified by ELISA (30 control and 59 AMD plasma) are shown with median (o) results ± first and third quartiles (Ql, Q3) and mean (Δ) results ± standard deviation (SD). P-values (two sided t-Test) were determined from log-transformed concentrations. Correlation between CML and pentosidine concentrations are shown for the control (E) and AMD (F) cohorts with horizontal and vertical dashed lines indicating median control values. Significantly more donors with both CML and pentosidine elevated are apparent in AMD patients than in the controls (upper right quadrants in E and F).

Figure 2. Correlation Between CML, Pentosidine and CEP Adducts in AMD Plasma. Plasma CEP adduct concentrations (A) and CEP autoantibody titers (B) quantified by ELISA from control (n = 30) and AMD donors (n = 56) are shown with median (o) results ± first and third quartiles (Ql, Q3) and mean (Δ) results ± standard deviation (SD). P-values (two sided t-Test) were determined from log-transformed concentrations. Correlation between CML and CEP adduct concentrations (C) and between pentosidine and CEP adduct concentrations (D) are shown with horizontal and vertical dashed lines indicating median control values. Odds ratios for AMD risk and 95% confidence intervals were determined by logistic regression based on two markers elevated relative to the median control values; p values were determined using the Fischer exact Test.

Figure 3. Plasma CML and Pentosidine by Donor Age. Plasma protein CML (A) and pentosidine (C) levels in the control (n = 30) and AMD (n = 60) cohorts are shown plotted by donor age. Pearson's correlation analysis revealed gradual increases with age for CML in both control and AMD donors and for pentosidine in AMD donors. Control donors exhibited little change in pentosidine levels with age. Plasma protein CML (B) and pentosidine (D) levels in control and AMD donors are plotted by age group, including 51-60 y (n = 4), 61-70 y (control, n = 12; AMD, n = 18), 71-81 y (control, n = 18; AMD, n = 28), > 81 y (n = 10). Fold differences in CML and pentosidine concentrations are indicated between control and AMD groups. Asterisks reflect p-values from a two sample t-Test (*** p < 0.001).

Figure 4. Plasma CML and Pentosidine Concentrations Stratified by Demographic and Health Factors. Plasma protein CML and pentosidine levels in the AMD and control cohorts are plotted based on donor status with regard to gender, status of smoking, hypertension, hyperlipidemia, diabetes and cardiovascular diseases. Sample size per group is indicated and asterisks reflect p- values from a two sample t-Test of log-transformed marker concentrations (*** p < 0.001, ** p < 0.01, * p < 0.05). F, female, M, male; S, smokers; NS, non-smoking; w, with; w/o, without.

Figure 5. Furosine in AMD and Control Plasma. Furosine concentrations quantified by amino acid analysis are shown with median (o) ± first and third quartiles (Ql, Q3) and mean (Δ) ± SD. Non-diabetic AMD and control plasma donors exhibited -20% lower mean furosine levels than diabetic AMD donors. P-values (two sided t-Test) were determined from log-transformed concentrations. Figure 6. A table of CML and pentosidine markers in control and AMD plasma.

Figure 7. A table of C-statistics for CML and pentosidine.

Figure 8. A table of sensitivity and specificity of CML, pentosidine and CEP adducts.

Figure 9. A table of the characteristics of the study population.

Figure 10. MS/MS spectra used to identify high intensity product ions for MRM monitoring of CML, pentosidine, argpyrimidine and the internal standard pyridylethyl-Cysteine (PEC). MS/MS conditions were adjusted to optimize the intensity of the parent ion and predominant product ion.

Figure 11. Representative Chromatography from LC System 1 of CML, argpyrimidine and pentosidine standards and internal standard PEC in the presence of plasma hydro lysate. Chromatography details are as described in experimental procedures.

Figure 12. Representative Chromatography of pentosidine and argyrimidine using LC system 2 and ammonium acetate/acetonitrile solvents with fluorescence detection at 335 nm excitation and 385 nm emission. Chromatography details are as described in Experimental Procedures.

Figure 13. Representative Chromatography of CML using LC system 2 and acetic acid/acetonitrile solvents and MRM at 205.1 / 84.1 m/z.

Figure 14. Representative Chromatography of PEC using LC system 2, ammonium acetate/acetonitrile solvents and MRM at 227.3 / 106.1 m/z.

Figure 15. Representative standard calibration curves using LC system 2 and MRM for CML and PEC and fluorescence monitoring for pentosidine. Each standard was analyzed at the indicated amounts in triplicate each day of analysis. Means + SD for each amount analyzed are shown. Figurel 6. Representative chromatography for quantification of furosine using AccQ Tag ™ amino acid analysis. Furosine peak 3, the putative di-derivatized form of the amino acid, was used for quantification of protein bound furosine. * Derivatization byproducts.

DETAILED DESCRIPTION

Age-related macular degeneration (AMD) causes severe vision loss in the elderly and early identification of AMD susceptibility could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we have evaluated the AMD predictive capability of plasma protein N^ε-carboxymethyllysine (CML) and pentosidine. CML and pentosidine are advanced glycation endproducts and abundant in Bruch's membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We quantified CML and pentosidine by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and LC-fluorimetry, respectively, and demonstrated -55% higher mean CML and -74% higher mean pentosidine concentrations in AMD (n = 60, p < 0.0001) relative to normal control plasma (n = 30). Plasma protein furosine concentrations were found by amino acid analysis to be the same in control and non-diabetic AMD donors, supporting an association between AMD pathology and increased levels of CML and pentosidine. Carboxyethylpyrrole (CEP) adducts, an oxidative modifications generated from docosahexaenoate-containing lipids and also abundant in AMD Bruch's membrane, were elevated - 2-fold in these AMD plasma but autoantibody titers to CEP, CML and pentodsidine were not significantly increased. Compelling higher mean levels of CML and pentosidine were found in AMD plasma over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control plasma donors with 80%, 81%, and 92% accuracy, respectively, while CML in combination with pentosidine provided -92% accuracy and CEP plus pentosidine provided - 95% accuracy. Demographic and health factor influences warrant further study but pentosidine levels appeared altered slightly in AMD patients with hypertension and cardiovascular disease. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD susceptibility, particularly in combination with concurrent analyses of furosine and CEP adducts. Elevated CML and Pentosidine in AMD Plasma. Fluorimetry and MRM tandem mass spectrometry coupled with two LC separations were used to quantify plasma protein-bound AGEs. MS/MS spectra used to identify high intensity product ions for MRM monitoring of CML, pentosidine, argpyrimidine and the PEC internal standard are shown in Fig. 10, available at MCP on line. Representative extracted ion and fluorescence LC profiles are shown for LC system 1 in Fig. 11 and for LC system 2 in Figs. 12-14. MRM standard calibration curves for CML and PEC and fluorescence calibration curves for pentosidine exhibited excellent linearity ( Fig 15).

Plasma from a total of 30 control subjects and 60 AMD patients were analyzed, including 30 early-stage dry AMD and 30 advanced-stage AMD patients. Protein was quantified by both PTC and AccQ-Tag amino acid analysis, yielding well defined protein concentrations with excellent agreement between the amino acid analysis methods (< 4% average difference). Amounts of CML and pentosidine in each HCl hydrolysate were corrected based on the percent recovery of the PEC internal standard which averaged 70.1 ± 12.4 (mean ± SD, n = 90). Overall, AMD patients exhibited -55% higher CML and -74% higher pentosidine concentrations relative to control plasma (Fig IAB and Figure 6 (Table I)). Comparison of log-transformed values confirmed the results with p < 0.0001 for both AGEs. Mean variability averaged -36% relative standard deviation (RSD) for CML and -24% RSD for pentosidine (n = 90). Relative to early stage AMD, advanced-stage AMD patients exhibited -10% higher CML (p = 0.30) and -20% higher pentosidine (p = 0.09), however these differences were not statistically significant (Figure 6 (Table I)). CML and pentosidine autoantibody titers measured by ELISA were -14-15% higher in AMD patients however these differences also were not statistically significant (Fig ICD).

Correlation of CML and pentosidine concentrations (Fig IEF) showed both markers to be elevated above median control levels in 87% of AMD patients (52/60) compared to only 23% of controls (7/30). Logistic regression modeling of the CML and pentosidine data yielded c- statistics of 0.80 and 0.92 respectively, for AMD patients (Figure 7 (Table 2)), supporting a 80% or 92% likelihood that a randomly selected person with elevated CML or pentosidine will be an AMD case rather than a control. Small difference in c-statistics were observed between early AMD and advanced-stage AMD with slightly higher values obtained for advanced stage disease. Bootstrap resampling and 10-fold cross-validation verified all c-statistics and 95% confidence intervals (CI) (Figure 7 (Table 2)). Risk of AMD was estimated by odds ratios (OR) for donors with elevated CML and pentosidine (Figure 6 (Table 2)). For all AMD, the data generated OR -9 for CML and OR ~11 for pentosidine; ORs for advanced AMD were greater than for early- stage disease. Each OR exhibited a large CI (Figure 6 (Table I)), indicating that these are preliminary estimates that lack precision.

Plasma CEP Λdducts and Autoantibodies in the Study Population. CEP plasma biomarkers offer potential utility is assessing AMD susceptibility (11), therefore it was of interest to compare CEP biomarkers with CML and pentosidine. In this study population, mean CEP adduct levels were ~ 2x higher (p < 0.0001) and mean CEP autoantibodies were -30% higher in AMD plasma (p = 0.25) (Fig 2AB). CEP autoantibody titers were higher than CML and pentosidine autoantibody titers in the AMD cohort but none were significantly elevated relative to the control cohort. Correlation of CEP adducts with CML and pentosidine (Fig 2CD) shows that all three oxidative protein modifications were elevated 75% - 77% above median control levels. Predicted risk of AMD yielded OR = 21.4 (95% CI, 6.9, 65.9) for pentosidine plus CML, OR = 9.9 (95% CI, 3.5, 27.9) for CML plus CEP adducts, and OR = 9.1 (95% CI, 3.3, 25.2) for pentosidine plus CEP adducts. Again the large confidence intervals indicate these OR estimates lack precision.

AMD Discriminatory Capability of Plasma CML, Pentosidine and CEP Adducts. Sensitivity and specificity measures were determined with receiver operating characteristic (ROC) curves from all AMD cases and controls for CML, pentosidine and CEP adducts alone, and for the combined markers (Figure 8 (Table 3)). Calculated to maximize the sum of the two values, sensitivity for CML (83%) was approximately the same as for pentosidine (85%) alone and greater than for CEP (68%) alone. The specificities of pentosidine (93%) and CEP (87%) alone were higher than that for CML alone (73%). The area under the ROC curve (c-statistic) is a measure of the overall discriminating accuracy of the markers and comparison of c-statistics suggested no significant difference in discriminatory accuracy between CEP (-81%) and CML (-80%) alone (p - 0.81) and between pentosidine and CEP alone (p - 0.10), however pentosidine exhibited significantly higher discrimination accuracy (-92%) than CML (p -0.03). C-statistics for the joint effect of combined markers were verified by bootstrap resampling and by 10-fold cross-validation. Comparison of c-statistics for combined markers suggested no significant difference in discriminatory accuracy between CEP plus CML (-89%) versus either marker alone (p - 0.1), nor between CEP plus pentosidine (-95%) versus pentosidine alone (p - 0.2) however CEP plus pentosidine appeared to be a better discriminator than CEP alone (p -0.02) and CML plus pentosidine (-92%) may be a better discriminator than CEP alone (p~ 0.06).

The Influence of Demographic and Health Factors on Plasma CML and Pentosidine. Study population characteristics are summarized in Figure 9 (Table 4), including age, gender, race, smoking status and health history. Comparisons by donor age revealed that CML concentrations increased gradually with age in both control and AMD donors (Fig 3A), and that AMD patients exhibit significantly higher mean levels of CML than controls at all ages (Fig 3B). Mean pentosidine increased gradually with age in AMD donors, remained stable with age in controls (Fig 3C) and was higher in AMD than in control plasma at all ages analyzed (Fig 3D).

A comparison of plasma protein CML and pentosidine concentrations by gender and health history is shown in Fig 5, including smoking, hypertension, hyperlipidemia, diabetes and cardiovascular disease. For each comparison, significant differences in CML and pentosidine concentrations were observed between AMD and control donors. The detection of differences within the AMD and control cohorts was limited by sample size, particularly for the control cohorts, and no significant differences in CML concentrations were detected within any of the cohorts. Small but significant differences were detected in pentosidine levels within two of the AMD cohorts. Specifically, mean pentosidine levels were higher in AMD pateints with hypertension and cardiovascular disease.

Because diabetes is such a prevalent disease, and because both CML (35, 36) and pentosidine (37, 38) have been reported to be elevated in diabetic plasma, we considered the possibility that undiagnosed diabetes in our study group contributed to the observed CML and pentosidine concentrations. For this purpose we quantified plasma protein furosine, a marker of early glycation, in all donor samples (Fig 5) as shown by the representative furosine amino acid analysis chromatograms in Fig 16. Protein quantification from the furosine analyses were in good agreement with previous amino acid analyses of these samples (average quantitative difference ~ 1.7%, n = 90). The average concentration of plasma protein furosine was 1.03 ± 0.11 pmol/μg in normal controls (n = 30), 1.03 ± 0.09 pmol/μg in non-diabetic AMD donors (n = 55), and 1.24 ± 0.05 pmol/μg in previously diagnosed diabetic AMD patients (n = 5). No difference was found in mean furosine concentrations between controls and non-diabetic AMD patients however the five diabetic AMD donors exhibited -20% higher furosine levels than the controls.

CML is a lysine modification and pentosidine is a fluorescent lysine-arginine crosslink, and both are formed through the ubiquitous Maillard reaction, which combines sugar carbonyls with primary amino groups to form glycated residues called Amadori products. Amadori products undergo subsequent reactions including intramolecular rearrangements and oxidative fragmentations to produces heterogeneous modifications collectively known as AGEs. Extracellular matrix proteins like collagen are particularly susceptible to AGE modification because of slow turnover rates and tissue and circulating AGE levels are higher in smokers and in those on a high AGE diet (18). AGE formation can lead to a myriad of effects, including altered protein function and activation of intracellular signaling pathways. Previous studies have implicated AGEs in the pathogenesis of AMD (4, 5, 7, 15, 16) and as well as other age- associated diseases, including atherosclerosis, arthritis, Alzheimer's disease, and diabetic complications (18, 35-41). Notably, AGEs formation and diabetic retinopathy have been reduced or prevented in a rodent model by treatment with pyrioximine (42), a derivative of vitamin B₆, and the risk of AMD in women (43) has been reduced by treatment with pyridoxine (vitamine B₆) in combination with folic acid and cyanocobalamin (vitamin Bi₂).

In the present study, we measured plasma protein CML and pentosidine in a study population of 90 subjects and demonstrated significantly higher mean CML and pentosidine concentrations in AMD plasma. Comparison of CML and pentosidine plasma concentrations between early-stage and advanced-stage AMD showed no significant differences, however analysis of larger cohorts might, especially for pentosidine (-20% difference, p -0.09). It was previously found (11) that mean CEP adducts were elevated -60% and mean CEP autoantibody titers elevated -30% (both with p < 0.0001) in a much larger study population (916 AMD and 488 control plasma). In this study population, mean CEP adducts were elevated 2x in the AMD cohort but CEP autoantibody titers were not significantly different than controls. CML and pentosidine autoantibodies also were not significantly elevated in this study population, suggesting that oxidative protein modifications may offer more promise as AMD biomarkers than autoantibodies.

This study found mean pentosidine concentrations elevated in the youngest AMD age group analyzed (i.e., 51-60 years) and both CML and pentosidine were elevated in plasma from those with early- stage dry AMD. Our preliminary analyses detected no confounding influences regarding plasma CML concentrations, however pentosidine levels were significantly higher in AMD donors with hypertension or cardiovascular disease. Elevated CEP adducts have also been detected in AMD plasma (11) however epidemiological studies have inconsistently associated hypertension and cardiovascular disease with AMD (48).

We quantified furosine because of the association of AGEs with diabetic complications and the prevalence of diabetes. Control and nondiabetic AMD plasma protein samples exhibited mean furosine concentrations of -1.0 pmol/μg protein, in excellent agreement with reported values for nondiabetic plasma, ie, 0.9-1.0 pmol furosine/μg protein (39-41). Except for five diagnosed diabetic AMD donors with mean furosine of 1.24 pmol/μg, no other donor sample in the study population exhibited furosine > 2 SD above the mean control level (ie, > 1.25 pmol/μg). Arguably, furosine concentrations within 1 SD of the mean diabetic level (ie, > 1.19 pmol/μg) might be suspect diabetics and 5 donor plasma in our study group fit this criteria. For these 3 control and 2 AMD patients, CML and pentosidine concentrations were < 1 SD from mean control or AMD level, respectively and medical records provided no indication that they were diabetics. These 5 plasma also did not exhibit significantly altered CEP adduct levels, consistent with our previous finding (11) that CEP adducts are not elevated in AMD diabetic plasma (determined from the analysis of 796 diabetic and 130 non-diabetic AMD plasma). Overall the furosine analyses showed no unknown diabetics were present in the study population and support an association between AMD pathology and increased levels of plasma CML and pentosidine. The present results compellingly demonstrated higher CML and pentosidine levels in AMD plasma proteins therefore their potential AMD biomarker efficacy was compared with each other and with CEP adducts. Odds ratios for AMD risk based on elevated CML (OR 9, 95% CI 3.0, 27.3) and pentosidine (OR 11, 95% CI 3.4, 35.2) for all AMD cases suggest that CML and pentosidine offer prognostic potential, however, the large confidence intervals indicate that more analyses are required to refine precision. Nevertheless, these OR estimates were greater than the OR based on elevated CEP adducts (OR 5.2, 95% CI 1.9, 14.3). For comparison, elevated CEP adducts and CEP autoantibodies combined yielded an OR of 3.17 (95% CI 2.51, 4.02) for AMD risk in a larger study population (n = 1404) (11). The area under the ROC curves (c-statistics) suggest that CML and CEP adducts alone can discriminate between AMD and control plasma donors with approximately equal accuracy (-80% - 81%) and that pentosidine alone can discriminate with higher 92% accuracy. C-statistics for the joint effect of markers were increased relative to those for the single markers but significant differences were only associated with pentosidine plus CEP, a combination that appeared to be a better discriminator of AMD than CEP alone (p -0.02), and possibly with CML plus pentosidine (-92%), a combination which also may be a better discriminator than CEP alone (p~ 0.06).

Pentosidine and CML are the most well studied AGEs, partly because of the availability of a variety of assays for their detection and quantification. Nevertheless, comparison of quantitative results across multiple studies remains complicated by variations in sample preparation, specimen storage, AGEs assay methods, protein quantification methods and by the array of formats used to report quantitative AGEs data (eg, pmol/ml, pmol/mg protein, fmol/nmol Lys, mmol/mol hydroxyproline, among others). In this study, the variability of the LC-MS and LC- fluorimetry measurements for CML and pentosidine (-36% and -24% RSD, respectively) was significantly lower than the variability of our ELISA measurements for CEP adducts (-50-52% RSDs) (11). The variability of our CML and pentosidine autoantibody results may be due in part to the plasma storage time (ie, 4-34 months). However, storage time (at -8O⁰C under argon with antioxidants) probably had little impact on the AGEs and protein measurements since protein was precipitated, washed and hydrolyzed immediately upon thawing the plasma. We quantified protein using two methods of amino acid analysis and obtained low variability (< 5% average RSD per sample) and excellent quantitative agreement (<4% average difference) among duplicate analyses per method.

Overall, this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD susceptibility, particularly in combination with CEP markers. The statistical analyses suggest that plasma levels of CML together with pentosidine discriminate between AMD and control patients with 92% accuracy and that pentosidine in combination with CEP adducts can discriminate with 95% accuracy. AGEs are systemic markers of inflammation and factors potentially confounding the use of CML and pentosidine in some cases. Monitoring furosine along with CML and pentosidine provides an effective method to narrow the causes of increased AGEs to possibly AMD susceptibility. When plasma furosine, CML and pentosidine are all elevated, an accurate clinical assessment will require more information. In such cases, monitoring CEP adducts could help rule out diabetic complications since plasma CEP adducts are elevated in AMD but not in diabetes.

EXPERIMENTAL PROCEDURES

Case-Control Study Design. Clinically documented AMD and control blood donors were recruited prospectively between 2005 and 2008 from the Cole Eye Institute, Cleveland Clinic Foundation with Institutional Review Board approval and according to Declaration of Helsinki principles. All patients received a comprehensive eye examination by a clinician in the Clinical Study Group and provided written informed consent. Human identifiers were removed and the blood specimens encoded by the Clinical Study Group to protect donor confidentiality. AMD disease progression was categorized based on fundus examination and patients were included in the study from AREDS AMD categories 2 and 4 (3). Briefly, AMD category 2 patients exhibited early- stage disease with multiple small drusen, single or nonextensive intermediate drusen (63- 124 μm), RPE pigmentary anormalities, or any combination of these, in one or both eyes and visual acuity of 20/30 or better in both eyes. AMD category 4 patients exhibited advanced AMD with substantial CNV or geographic atrophy involving the macula in one or both eyes. Control donors lacked macular drusen and exhibited no clinical evidence of any retinal disorder. Human Plasma Preparation. Nonfasting blood specimens were collected in BD Vacutainer^® K₂EDTA tubes and plasma was prepared within 6 hours and aliquotted to vials containing the antioxidant butylated hydroxytoluene (BHT; 1 mg/ml plasma) and a protease inhibitor cocktail (Sigma product number P 8340; lOμl/ml plasma) (11). The plasma was flushed with argon, quench-frozen in liquid nitrogen immediately and stored at - 80 ⁰C until analysis. For the plasma used in this study storage time at - 80 ⁰C ranged from 4-34 months and averaged 13 months. All samples were frozen and thawed only once.

Sample Preparation and Amino Acid Analysis. Plasma (-200 μl, —10 mg) was transferred to 6 x 50 mm glass hydrolysis tubes and protein was precipitated with 2 volumes of cold acetone. After incubation at 4⁰C for 10-20 min, the preparation was centrifuged briefly on a microfuge, the supernatant discarded and the pellet washed once with 67% acetone (400 μl) and vacuum dried (32). Plasma protein was prepared for hydrolysis by adding 60 μl of 6 N HCl to each dried pellet then the hydrolysis tubes were placed in a 40 ml screw-cap vial containing -300 μl of 6N HCl with a few small crystals of phenol. The 40 ml vial was capped with a mininert slide valve, the valve was connected to a vacuum pump and argon source via a three-way stopcock and the vial alternately evacuated and flushed with argon 3x then sealed under vacuum (32). Protein was hydro lyzed at HO⁰C for 16 h, then vacuumed dried, flushed with argon and stored at -2O⁰C until analysis. Protein was quantified by PTC amino acid analysis (-80 μg derivatized and -3 μg analyzed) using an Agilent 1100 HPLC system, a Haisil PTC C18 column (220 x 2.1 mm, Applied Biosytems), and a Gilson model 116 UV detector (32). Protein was also quantified by AccQ-Tag™ amino acid analysis (-3.5 μg derivatized and -35 ng analyzed) using an Acquity Ultra Performance LC system (Waters) and AccQ-Tag Ultra column (100 x 2.1 mm) according to the vendor (33). Bovine serum albumin from the National Bureau of Standard was used as a protein standard and hydrolysis control. Amino acid calibration standards were obtained from Pierce and Thermo Scientific.

Furosine Quantification. Furosine [ -N-(2-furoylmethyl) lysine] was quantified by duplicate AccQ'Tag™ amino acid analyses (-15 μg derivatized and -2.7 μg analyzed) using the Acquity LC system (Waters) described above. Furosine contains a primary and a secondary amino group and both are derivatized by the AccQ'Tag™ reagent, yielding two different mono-derivatized forms and a di-derivatized species. The apparent di-derivatized species was well separated from other amino acids, exhibited a constant response factor up to ~55 pmol derivatized and was used for quantification of furosine in protein hydrolysates. Furosine standard was purchased from NeoMPS, Inc.

CML and Pentosidine Quantification. Plasma protein HCl hydrolysates (~8 mg in 40 μl H₂O) were spiked with a PEC internal standard (15 pmol) and fractionated on LC system 1 composed of an Agilent 1100 HPLC, a Hypercarb™ porous graphite carbon column (5 μm particles, 50 x 10 mm, Thermo Scientific) maintained at 30⁰C with an Applied Biosytems 112A column oven, aqueous trifluoroacetic acid/acetonitrile solvents, and using gradient elution (0-100% acetonitrile over 13 min) and a flow rate of 1 ml/min. The eluent was monitored for fluorescence ( 335 nm excitation, 385 nm emission) with a Waters™ 474 scanning fluorimeter and initially split with 20% directed to an API 3000 triple quadrupole electrospray mass spectrometer (Applied Biosystem) and 80% to a fraction collector. After determining reproducible elution times using control plasma spiked with standard AGEs and PEC, 100% of the eluant was directed to the fraction collector and three fractions were collected, one each for CML, PEC, and the co-eluting pentosidine plus argpyrimidine. The fractions were vacuum dried and re-fractionated on LC system 2 composed of the same HPLC equipment but with an aqueous normal phase Cogent Diamond Hydride™ Column (4.2 μm particles, 150 x 2.1 mm) used at room temperature. Fractions containing CML were re-chromatographed using aqueous acetic acid/acetonitrile solvents and gradient elution (95%-0% acetonitrile in 13.6 min) at a flow rate of 400 μl/min. Fractions containing PEC, argpyrimidine and pentosidine were re-chromatographed using 10 mM ammonium acetate, pH 6.0/acetonitrile solvents, gradient elution (100%-0% acetonitrile in 15 min) at a flow rate of 400 μl/min. Aqueous normal phase chromatography was monitored by fluorescence detection followed by 100% of the eluant directed to the mass spectrometer. CML and PEC were quantified by multiple reaction monitoring (MRM) and pentosidine and argpyrimidine were measured by fluorescence; final CML and pentosidine amounts were adjusted based on the recovery of the PEC internal standard. Plasma protein argpyrimidine concentrations were below reliable detection limits in this analytical system and not reported. Calibration curves were developed in triplicate each day of analysis using LC system 2 and external standards. CML standard was purchased from NeoMPS, Inc., pentosidine was obtained from the International Maillard Reaction Society (Case Western Reserve University, Cleveland, OH), argpyrimidine was prepared in our laboratories by RN, and s-β(4-pyridylethyl)-L-cysteine (PEC) was purchased from Sigma.

Mass Spectrometry. The mass spectrometer was operated with Analyst 1.3.1 software (Applied Biosytems) and MS/MS spectra were generated on singly charged precursor ions for CML, pentosidine, argpyrimidine and PEC and specific transition ions for each modified amino acid were analyzed by MRM. The declustering potential, focusing potential, collision energy, and exit potential were optimized for each ion to ± 0.1 Da and ± 1 volts. Ion spray voltage was set at 5300 V and source temperature at 425°C in LC system 1 and at 490⁰C in LC system 2. The m/z of the precursor ions and each of the transitions ions and their optimized voltages were transcribed respectively into an LCsync method in the Analyst 1.3.1 software. CML was quantified by MRM using precursor ion 205.1 and product ion 84.1; PEC was quantified using precursor ion 227.3 and product ion 106.1; pentosidine was monitored by MRM using precursor ion 379.2 and product ion 187.2; and argpyrimidine was monitored using precursor ion 255.2 and product ion 237.3. Peak areas and external standard calibration curves were used for quantification of CML and PEC by MRM and pentosidine by fluorescence.

Pentosidine and CML Autoantibody Assays. Pentosidine autoantibody titers were measured by direct ELISA using pentosidine modified bovine serum albumin (pentosidine-BSA) and unmodified BSA as coating antigen (~8 μg/well). CML autoantibody titers were measured with the same methodology using CML-BSA and BSA as coating agent (~15 μg/well). Plasma (100 μl of a 1 :10 dilution) was applied to coating agents and the ELISA developed as previously described for the CEP autoantibody assay (9, 11). Anti-CML (R&D Systems, product MAB3247) or anti-pentosidine monoclonal antibodies (Trans Genie Inc, Japan, product KHO 12) were used for positive ELISA controls. Titer was defined as the ratio of plasma binding to antigen (A) versus binding to BSA (A₀) (9).

Preparation of AGEs Modified BSA. Pentosidine-BSA was prepared by mixing pentosidine (1 mg, NeoMPS, product SC1535) in dimethylformamide (100 μl) with l-ethyl-3-(3- dimethylaminopropyl) carbodiimide HCl (5 mg, Pierce) and N-hydroxysuccinimide (6 mg, Pierce) at room temperature for 2 hours followed by the addition of BSA (2.5 mg in 0.5 ml PBS, Sigma product A6003) and continued incubation at 37⁰C for 48 h. The reaction was stopped by dialysis against 25 mM Tris-HCL (pH 8.0) for 2 hours at 4⁰C, then dialyzed against PBS for another 24 h with two changes. CML-BSA was prepared by mixing BSA in PBS with 25 mM glyoxylic acid and 50 mM NaCNBH₃ for 48 h at room temperature followed by dialysis against PBS for 24 h with two changes (34). Protein was quantified by the BCA assay (Pierce) and pentosidine and CML modifications confirmed by Western analysis before and after the modification reactions using anti-CML monoclonal antibody (R&D Systems, product MAB3247) or anti-pentosidine monoclonal antibody (Trans Genie Inc, Japan, product KHO 12).

CEP Adduct and CEP Autoantibody Assays . The CEP adduct and autoantibody concentrations of plasma used in this study were previously reported among 1404 plasma (11). Briefly, CEP adducts were quantified with a competitive ELISA using rabbit anti-CEP polyclonal antibody, CEP modified bovine serum albumin as coating agent and known amounts of CEP modified human serum albumin as reference protein. CEP autoantibody titers were measured by direct ELISA using CEP-BSA as coating antigen. These methods are well documented (9, 11).

Statistics. Continuous measures were summarized using means, standard deviations, medians and interquartile ranges, while categorical factors were described using frequencies and percentages. Differences between control and AMD patients in plasma concentrations of CML, pentosidine, and CEP adducts as well as in CML, pentosidine, and CEP autoantibodies were evaluated using two sample t-Tests in Excel 2003 (Microsoft^® Office). To evaluate a relationship between CML and pentosidine with AMD susceptibility, a logistic regression model was fit with both variables as predictors using SAS 9.1 software (SAS Institute, Cary, NC). C-statistics measured the model's ability to discriminate between AMD and controls, and odds ratios (ORs) showed the change in risk of AMD based on the predictors. ORs, c-statistics and p-values were determined based on log-transformed marker concentrations. Validation of c-statistics was performed using 2000 bootstrap (random) resamplings to calculate empirical 95% confidence intervals (CI) and by performing 10-fold cross-validation. Sensitivity and specificity were calculated to maximize the sum of the two values using receiver operating characteristic (ROC) curves constructed with SAS 9.1 from the output of logistic regression analysis fit with either CML, pentosidine or CEP adducts alone or in combination. C-statistics and p-values comparing ROC curves were determined with SAS 9.1. For association analyses of combined effects of CML and pentosidine with CEP adducts, ORs with 95% CI and Fisher Exact p-values were calculated with SAS 9.1 software. Pearson's correlation analysis in Minitab Release 15 (Minitab Inc.) was used to compare concentrations of AGE markers with plasma donor age.

Abbreviations. AGEs, advanced glycation end-products; AMD, age-related macular degeneration; AccQ, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; AREDS, age-related eye disease study; CEP, 2-(ω-carboxyethyl)pyrrole; CI, confidence interval; CML, N^ε- (carboxymethyl)lysine; CNV, choroidal neovascularization; OR, odds ratio; PEC, s-β(4- pyridylethyl)-L-cysteine; PTC, phenylthiocarbamyl; ROC, receiver operating characteristic; RPE, retinal pigment epithelium; RSD, relative standard deviation; SD, standard deviation.

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WHAT IS CLAIMED IS:

Claims

WHAT IS CLAIMED IS:

1. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level N(6)-carboxymethyllysine (CML) in a bodily fluid of the subject;

(b) comparing the level of CML measured to a control value; and

(c) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the control value or characterizing the subject as at lesser risk of developing AMD if the level of CML measured is not greater than the control value.

2. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a control value;

3. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level of at pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) measuring the level of CML in a bodily fluid of the subject;

(d) comparing the level CML measured to a CML control value; and

4. A method for assessing the risk that a subject is will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the of level N(6)-carboxymethyllysine (CML) in a bodily fluid of the subject;

(b) comparing the level of CML measured to a CML control value;

(c) measuring the level of a 2-( -carboxyethyl)pyrrole adduct (CEP adduct) or anti-CEP antibodies in a bodily fluid of the subject;

(d) comparing the level of CEP adduct or anti-CEP antibodies measured to a CEP adduct control value; and

(f) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the CML control value and the level of CEP adduct or anti- CEP antibodies measured is greater than the CEP adduct control value or characterizing the subject as at lesser risk of developing AMD if the level of CML measured is not greater than the CML control value and the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value.

5. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) measuring the level 2-( -carboxyethyl)pyrrole (CEP) or anti-CEP antibodies in a bodily fluid of the subject;

(f) characterizing the subject as at greater risk of developing AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adduct or anti-CEP antibodies measured is greater than the CEP adduct control value or characterizing the subject as at lesser risk of developing AMD if the level of pentosidine measured is not greater than the pentosidine control value and the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value.

6. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(c) comparing the level of CML measured to a CML control value; (d) measuring the level 2-(ω-carboxyethyl)pyrrole adduct (CEP adduct) or anti-CEP antibodies in a bodily fluid of the subject;

(e) comparing the level of CEP adduct or anti-CEP antibodies measured to a CEP adduct control value; (f) measuring the level of pentosidine in a bodily fluid of the subject;

(g) comparing the level of pentosidine measured to a pentosidine control value; and

(h) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the CML control value, the level of CEP adduct or anti-CEP antibodies measured is greater than the CEP adduct control value, and the level of pentosidine measured is greater than the pentosidine control value or characterizing the subject as at lesser risk of developing AMD if the level of CML measured is not greater than the CML control value, the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value, and the level of pentosidine measured is not greater than the pentosidine control value.

7. The method of any of claims 1, 3, 4 and 6 wherein the step of measuring CML in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.

8. The method of any of claims 2, 3, 5 and 6 wherein the step of measuring pentosidine in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.

9. The method of any of claims 4, 5 and 6 wherein the step of measuring CEP adduct or anti-CEP antibody in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.

10. The method of claim 7 wherein the bodily fluid is selected from serum and plasma.

11. The method of claim 8 wherein the bodily fluid is selected from serum and plasma.

12. The method of claim 9 wherein the bodily fluid is selected from serum and plasma.

13. The method of any of claims 1 -6 wherein the subject is not suffering from diabetes.

14. The method of claim 4 further comprising characterizing the subject as at lesser risk of developing AMD if the level of CML measured is greater than the CML control value and the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value.

15. The method of claim 5 further comprising characterizing the subject as at lesser risk of developing AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value.

16. The method of claim 6 further comprising characterizing the subject as at lesser risk of developing AMD if the level of CML measured is greater than the CML control value, the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value, and the level of pentosidine measured is greater than the pentosidine control value.

17. The method of any of claims 1-6 wherein the AMD is advanced AMD and the control value is a control value for mild to moderate AMD.

18. The method of any of claims 1-6 wherein the AMD is mild to moderate AMD and the control value is a control value for non-AMD.

19. The method of any of claims 1 -6 wherein the AMD is dry AMD.

20. The method of any of claims 1 -6 wherein the AMD is wet AMD.

21. The method any of claims 1-6 further comprising obtaining a genetic sample from the subject and determining whether the subject has a genetic marker associated with increased risk for developing AMD, wherein the genetic marker is that associated with the risk allele at a SNP selected from the group consisting of: rsl0737680 (CHF), rs3793917 (ARMS2/HTRA1), rs429608 (C2/CFB), rs2230199 (C3), rs2285714 (CFH), rs9380272 (C2/CFB), rs9621532 (SYN3/TIMP3), rs493258 (LIPC), rs3764261 (CETP), rsl2678919 (LPL), and rsl883025 (ABCAl), wherein the method comprising assessing the patient is at increase risk for developing AMD if the subject has a genetic marker associate with the risk allele. .

22. The method of any of claims 1-6 wherein the characterizing step comprises characterizing the subject as having a less than an 60% chance of developing AMD.

23. The method of any of claims 1-6 wherein the characterizing step comprises characterizing the subject as having a greater than an 60% chance of developing AMD.

24. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level N(6)-carboxymethyllysine (CML) in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound;

(d) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if the increase in the level of CML measured is less than the CML control value increase.

25. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound; (b) comparing the increase in the level of pentosidine measured between the first and second time to a pentosidine control value increase; and

26. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level N(6)-carboxymethyllysine (CML) in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(c) measuring the level of CEP adduct or anti-CEP antibodies in the serum of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(d) comparing the increase in the level of CEP adduct or anti-CEP antibodies measured between the first and second time to a CEP adduct control value increase; and

(e) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of CEP adduct or anti-CEP antibodies measured is less than the CEP adduct control value increase.

27. The method of claim 26 wherein step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of CEP adduct or anti-CEP antibodies measured is less than the CEP adduct control value increase.

28. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

29. The method of claim 28 wherein step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of CML measured is less than the CML control value increase and (ii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.

30. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily of a subject at a first time before administering the test compound and at a second time after administering the test compound;

(b) comparing the increase in the level of pentosidine measured between the first and second time to a pentosidine control value increase; (c) measuring the level of CEP adduct or or anti-CEP antibodies in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(f) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of pentosidine measured is less than the pentosidine control value increase and (ii) the increase in the level of CEP adduct or anti-CEP antibodies measured is less than the CEP adduct control value increase.

31. The method of claim 30 wherein step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the increase in the level of pentosidine measured is less than the pentodine control value increase and (ii) the increase in the level of CEP adduct or anti-CEP adduct antibodies measured is less than the CEP adduct control value increase.

32. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(c) measuring the level CEP adduct or anti-CEP antibody in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(d) comparing the increase in the level of CEP adduct or anti-CEP antibodies measured between the first and second time to a CEP adduct control value increase; and (e) measuring the level pentosidine in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound; (f) comparing the increase in the level of pentoside measured between the first and second time to a pentoside control value increase; and

(g) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adduct or anti-CEP antibodies measured is less than the CEP adduct control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase

33. The method of claim 32 wherein step (g) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if two or more of: (i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adduct or anti-CEP antibodies measured is less than the CEP adduct control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.

34. The method of claim 32 wherein step (g) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if :(i) the increase in the level of CML measured is less than the CML control value increase; (ii) the increase in the level of CEP adduct or anti-CEP antibodies measured is less than the CEP adduct control value increase; and (iii) the increase in the level of pentosidine measured is less than the pentosidine control value increase.

35. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level N(6)-carboxymethyllysine (CML) in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound; and

(b) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

36. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily fluid of a subject at a first time before administering the test compound and at a second time after administering the test compound;

37. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(b) measuring the level CEP adduct or anti-CEP antibodiesin the serum of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(c) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of CEP adduct or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound. and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

38. The method of claim 37 wherein step (c) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both: (i) the level of CEP adduct or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

39. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(b) measuring the level pentosidine in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

40. The method of claim 30 wherein step (e) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound.

41. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily of a subject at a first time before administering the test compound and at a second time after administering the test compound; (b) measuring the level CEP adduct or anti-CEP antibodies in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound; (c) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CEP adduct or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

42. The method of claim 41 wherein step (c) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if both (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound; and (ii) the level of CEP adduct or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

43. A method for identifying a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD), the method comprising:

(b) measuring the level of CEP adduct or anti-CEP antibodies in a bodily fluid of the subject at a first time before administering the test compound and at a second time after administering the test compound;

(d) identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if one or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adduct or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

44. The method of claim 43 wherein step (d) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if two or more of: (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adduct or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound

45. The method of claim 43 wherein step (d) comprises identifying the test compound as a candidate compound for treating or reducing the risk of developing age-related macular degeneration (AMD) if (i) the level of pentosidine measured after administering the test compound is less than the level measured before administering the test compound and (ii) the level of CML measured after administering the test compound is less than the level measured before administering the test compound; and (iii) the level of CEP adduct or anti-CEP antibodies measured after administering the test compound is less than the level measured before administering the test compound.

46. The method of any of the forgoing claims wherein the step of measuring comprises the use of mass spectrometry.

47. The method of any of the forgoing claims wherein the bodily fluid is obtained from a biological sample obtained from the subject.

48. The method of any of claims 24-47 wherein the subject is a human taking part in a clinical trial.

49. The method of any of claims 24-47 wherein the subject is a non-human mammal.

50. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(b) comparing the level of CML measured to a control value; and

(c) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the control value.

51. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a control value;

(c) characterizing the subject as at greater risk of developing AMD if the level of pentosidine measured is greater than the control value.

52. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level of at pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) measuring the level of at CML in a bodily fluid of the subject;

(d) comparing the level CML measured to a CML control value; and

(e) characterizing the subject as at greater risk of developing AMD if the level of pentosidine measured is greater than the control value and the level of CML is greater than the CML control value

53. A method for characterizing the risk that a subject is will develop age-related macular degeneration (AMD), the method comprising:

(b) comparing the level of CML measured to a CML control value; (c) measuring the level 2-(ω-carboxyethyl)pyrrole (CEP adduct) or anti-CEP antibodies in a bodily fluid of the subject;

(f) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the CML control value and the level of CEP adduct or anti-CEP antibodies is greater than the CEP adduct control value

54. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(a) measuring the level pentosidine in a bodily fluid of the subject;

(b) comparing the level of pentosidine measured to a pentosidine control value;

(c) measuring the level 2-(ω-carboxyethyl)pyrrole (CEP adduct) or anti-CEP antibodies in a bodily fluid of the subject;

(f) characterizing the subject as at greater risk of developing AMD if the level of pentosidien measured is greater than the pentosidine control value and the level of CEP adduct or anti-CEP antibodies is greater than the CEP adduct control value

55. A method for characterizing the risk that a subject will develop age-related macular degeneration (AMD), the method comprising:

(c) comparing the level of CML measured to a CML control value;

(d) measuring the level 2-( -carboxyethyl)pyrrole (CEP adduct) or anti-CEP antibodies in a bodily fluid of the subject;

(e) comparing the level of CEP adduct or anti-CEP antibodies measured to a CEP adduct control value;

(f) measuring the level of pentosidine in a bodily fluid of the subject;

(g) comparing the level of pentosidine measured to a pentosidine control value; and (h) characterizing the subject as at greater risk of developing AMD if the level of CML measured is greater than the CML control value, the level of pentosidine is greater than the pentosidine control value, and the level of CEP adduct or anti-CEP antibodies is greater than the CEP adduct control value

56. The method of any of claims 50, 52, 53 and 55 wherein the step of measuring CML in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.

57. The method of any of claims 51, 52, 54 and 55 wherein the step of measuring pentosidine in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.

58. The method of any of claims 53, 54 and 55 wherein the step of measuring CEP adduct or or anti-CEP antibodies in a bodily fluid comprising obtaining a biological sample from the subject comprising a bodily fluid.

59. The method of claim 56 wherein the bodily fluid is selected from serum and plasma.

60. The method of claim 57 wherein the bodily fluid is selected from serum and plasma.

61. The method of claim 58 wherein the bodily fluid is selected from serum and plasma.

62. The method of any of the forgoing claims wherein the subject is not suffering from diabetes.

63. The method of claim 53 wherein the patient is diagnosed as not suffering from AMD if the level of CML measured is greater than the CML control value and the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value.

64. The method of claim 54 wherein the patient is diagnosed as not suffering from AMD if the level of pentosidine measured is greater than the pentosidine control value and the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP adduct control value.

65. The method of claim 55 wherein the patient is diagnosed as not suffering from AMD if the level of CML measured is greater than the CML control value, the level of CEP adduct or anti-CEP antibodies measured is not greater than the CEP control value, and the level of pentosidine measured is greater than the pentosidine control value.