Increased damage to proteins by glycation, oxidation and nitration has been implicated in neuronal cell death leading to Alzheimer's disease (AD). Protein glycation, oxidation and nitration adducts are consequently formed. Quantitative screening of these adducts in CSF may provide a biochemical indicator for the diagnosis of AD. To assess this, we measured 11 glycation adducts, three oxidation adducts and a nitration adduct, determining both protein adduct residues and free adducts, in CSF samples of age-matched normal healthy subjects (n ¼ 18) and subjects with Alzheimer's disease (n ¼ 32). In CSF protein, the concentrations of 3-nitrotyrosine, N e -carboxymethyl-lysine, 3-deoxyglucosone-derived hydroimidazolone and N-formylkynurenine residues were increased in subjects with Alzheimer's disease. In CSF ultrafiltrate, the concentrations of 3-nitrotyrosine, methylglyoxal-derived hydroimidazolone and glyoxal-derived hydroimidazolone free adducts were also increased. The Mini-Mental State Examination (MMSE) score correlated negatively with 3-nitrotyrosine residue concentration (p < 0.05), and the negative correlation with fructosyl-lysine residues just failed to reach significance (p ¼ 0.052). Multiple linear regression gave a regression model of the MMSE score on 3-nitrotyrosine, fructosyl-lysine and N e -carboxyethyl-lysine residues with p-values of 0.021, 0.031 and 0.052, respectively. These findings indicate that protein glycation, oxidation and nitration adduct residues and free adducts were increased in the CSF of subjects with Alzheimer's disease. A combination of nitration and glycation adduct estimates of CSF may provide an indicator for the diagnosis of Alzheimer's disease. Keywords: Alzheimer's disease, cerebrospinal fluid, glycation, methylglyoxal, nitrotyrosine, oxidative stress. Alzheimer's disease (AD) is characterized by the formation of extracellular protein deposits consisting predominantly of amyloid peptide and intracellular protein deposits of the microtubule-associated protein tau. These are found in the neocortex, entorhinal cortex, and hippocampus. Advanced glycation endproduct (AGE) residues accumulate in the deposited proteins. This contributes to their insolubility and protease resistance. Neuronal damage in AD is also associated with increased protein oxidation and nitration. These Abbreviations used: AGEs, advanced glycation endproducts; AD, Alzheimer's disease; CEL, N e -carboxyethyl-lysine; CML, N e -carboxymethyl-lysine; 3DG-H, N d -(5-hydro-5-(2,3,4-trihydroxybutyl)-4-imidazolon-2-yl)ornithine and related structural isomers; DOLD, 3-deoxyglucosone-derived lysine dimer; FL, fructosyl-lysine; G-H1, N d -(5-hydro-4-imidazolon-2-yl)ornithine; GOLD, glyoxal-derived lysine dimer; LC-MS/MS, liquid chromatography with triple quadrupole mass spectrometric detection; LOD, limit of detection; MG-H1, N d -(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine; MetSO, methionine sulfoxide; MMSE, Mini-Mental State Examination; MOLD, methylglyoxal-derived lysine dimer; MRM, multiple reaction monit...

There is currently no biochemical test for detection of early-stage osteoarthritis (eOA). Tests for early-stage rheumatoid arthritis (eRA) such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibodies require refinement to improve clinical utility. We developed robust mass spectrometric methods to quantify citrullinated protein (CP) and free hydroxyproline in body fluids. We detected CP in the plasma of healthy subjects and surprisingly found that CP was increased in both patients with eOA and eRA whereas anti-CCP antibodies were predominantly present in eRA. A 4-class diagnostic algorithm combining plasma/serum CP, anti-CCP antibody and hydroxyproline applied to a cohort gave specific and sensitive detection and discrimination of eOA, eRA, other non-RA inflammatory joint diseases and good skeletal health. This provides a first-in-class plasma/serum-based biochemical assay for diagnosis and type discrimination of early-stage arthritis to facilitate improved treatment and patient outcomes, exploiting citrullinated protein and related differential autoimmunity.

BackgroundThere is currently no blood-based test for detection of early-stage osteoarthritis (OA) and the anti-cyclic citrullinated peptide (CCP) antibody test for rheumatoid arthritis (RA) has relatively low sensitivity for early-stage disease. Morbidity in arthritis could be markedly decreased if early-stage arthritis could be routinely detected and classified by clinical chemistry test. We hypothesised that damage to proteins of the joint by oxidation, nitration and glycation, and with signatures released in plasma as oxidized, nitrated and glycated amino acids may facilitate early-stage diagnosis and typing of arthritis.MethodsPatients with knee joint early-stage and advanced OA and RA or other inflammatory joint disease (non-RA) and healthy subjects with good skeletal health were recruited for the study (n = 225). Plasma/serum and synovial fluid was analysed for oxidized, nitrated and glycated proteins and amino acids by quantitative liquid chromatography-tandem mass spectrometry. Data-driven machine learning methods were employed to explore diagnostic utility of the measurements for detection and classifying early-stage OA and RA, non-RA and good skeletal health with training set and independent test set cohorts.ResultsGlycated, oxidized and nitrated proteins and amino acids were detected in synovial fluid and plasma of arthritic patients with characteristic patterns found in early and advanced OA and RA, and non-RA, with respect to healthy controls. In early-stage disease, two algorithms for consecutive use in diagnosis were developed: (1) disease versus healthy control, and (2) classification as OA, RA and non-RA. The algorithms featured 10 damaged amino acids in plasma, hydroxyproline and anti-CCP antibody status. Sensitivities/specificities were: (1) good skeletal health, 0.92/0.91; (2) early-stage OA, 0.92/0.90; early-stage RA, 0.80/0.78; and non-RA, 0.70/0.65 (training set). These were confirmed in independent test set validation. Damaged amino acids increased further in severe and advanced OA and RA.ConclusionsOxidized, nitrated and glycated amino acids combined with hydroxyproline and anti-CCP antibody status provided a plasma-based biochemical test of relatively high sensitivity and specificity for early-stage diagnosis and typing of arthritic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1154-3) contains supplementary material, which is available to authorized users.