Nitrosative stress where nitrosylation of tyrosine (Tyr) leading to 3-nitrotyrosine proteins or free 3-nitrotyrosine is the most prominent change has been proposed as a pathogenic mechanism in Parkinson’s disease (PD). amines are due to excess of nitric oxide. Nitrosylation of tyrosine (Tyr) leading to 3-nitrotyrosine proteins or free 3-nitrotyrosine is the most prominent change. Excess nitrosylation is recognized as a salient feature of α-synucleinopathies such as PD where 3-nitrotyrosine proteins such as nitrosylated neurofilaments and α-synuclein (αSyn) are detected in brain aggregates (5). However levels of 3-nitrotyrosine proteins or free 3-nitrotyrosine in serum and cerebrospinal fluid (CSF) of patients with PD have not been studied and the presence of free 3-nitrotyrosine deserves investigation because it is known to be neurotoxic for striatal neurons (7). Regarding αSyn this protein represents a main component of Lewy bodies the hallmarks of PD and mutations in αhave been linked to familial PD. This protein contains 140 amino acids with four tyrosine residues (Tyr39 at amine terminus and Tyr125 Tyr133 and Tyr136 at carboxyl terminus) which are readily accessible for modification by nitrating brokers. These residues can be differentially nitrosylated leading to different functional effects (4). Although parkinsonian nitrosylation of αSyn is usually suspected to occur in brain tissue this protein can also be detected in CSF BMS-540215 and blood (2). The presence of αSyn in CSF is already known but αSyn nitrosylation has not been studied. In contrast nitro-α-synuclein (N-αSyn) has been detected in blood mononuclear cells of patients with PD (9) although serum levels are not known. The objectives of this study were (i) to determine in serum and CSF of patients with PD and control subjects the presence of 3-nitrotyrosine proteins and free 3-nitrotyrosine markers for protein and amine nitrosylation and (ii) to discern the effects of nitrosylation BMS-540215 on serum and CSF αSyn. Development We report for the first time the presence of selective nitrosylation stress in serum and cerebrospinal fluid of patients with early Parkinson’s disease (PD) characterized by excess of 3-nitrotyrosine proteins other than nitroalbumin without free 3-nitrotyrosine. We also detected nitrosative changes in serum nitro-α-synuclein (N-αSyn) characterized by an altered profile of tyrosine (Tyr) nitrosylation. Thus the intensity of nitrosylation of Tyr125 136 residues is usually enhanced and that of the Tyr39 site is usually reduced and the ratio between both parameters is usually higher in patients with early PD relative to controls. Our observations lead to the hypothesis that evaluating nitrosative stress through enhanced levels of 3-nitrotyrosine proteins other than nitroalbumin together with the profile of tyrosine nitrosylation of serum N-αSyn could serve for the diagnosis of sporadic PD. BMS-540215 Results and Discussion First we verified that the main clinical characteristics were similar between the PD and control groups except for hypertension which was more frequent in patients (controls; Fig. 1). Regarding nitroalbumin the differences were not found to be significant (PD patients=0.09±0.02?μg/ml; controls=0.02±0.01?μg/ml). The Tibbling-Link index was lower than 0.7 in every patient with PD discarding that this increase in CSF BMS-540215 3-nitrotyrosine proteins was caused by enhanced blood levels. Since nitrosylative stress can also yield free nitrosylated amines such as 3-nitrotyrosine (7) we evaluated its presence in serum and CSF with mass spectrometry. 3-nitrotyrosine (retention time=9.19 IL-11 to 9.59?min) was not detected in any patient and only several unknown compounds with different retention times were observed in the MS spectra (Fig. 2). FIG. 2. Mass spectrometry (MS) spectra of 3-nitrotyrosine standard 1ppm and representative MS spectra of serum and CSF of a patient with PD. The transitions for nitrotyrosine are 227.1/181.0 227.1 and 227.1/116.9 with retention times from 9.19 to 9.59?min. … The findings suggested that nitrosylation was quite selective affecting 3-nitrotyrosine proteins other than albumin. Then we decided to evaluate which proteins were BMS-540215 nitrosylated. For this purpose immunoblots of serum needs previous depletion of serum albumin or immunoprecipitation of proteins of.