Plasma 3-nitrotyrosine is a biomarker in animal models of arthritis: Pharmacological dissection of iNOS' role in disease.

The contribution of inducible nitric oxide synthase (iNOS) to oxidative/nitrative stress is well-documented in inflammation, but difficult to quantify. Using a novel, recently developed assay for 3-nitrotyrosine (3-NT), we characterized iNOS activity and its inhibition in preclinical models of inflammation. In particular, we utilized the 3-NT assay to assess the role of iNOS in the disease pathology as well as for proof of pharmacology of iNOS inhibitors in an acute endotoxin challenge model, in models of rheumatoid arthritis (RA) such as rat adjuvant- and collagen-induced arthritis (AIA and CIA) and a model of osteoarthritis (OA) such as rat sodium monoiodoacetate-induced arthritis (MIA). Quantification of nitrotyrosine was performed using immuno-affinity 2-D LC-MS/MS assay. This assay is a very specific and reproducible and is amenable to a number of biological fluids. Plasma levels of 3-NT were significantly elevated in an acute model of inflammation (rat LPS) and in models of rheumatoid arthritis (adjuvant- and collagen-induced arthritis), and osteoarthritis (monoiodoacetate-induced arthritis). Plasma 3-NT correlated with the severity of the inflammatory response; thus, a 20-fold increase was observed in the rat LPS model, a 10-fold increase in AIA, and only a 2.5-fold elevation in CIA. Pharmacological intervention with iNOS inhibitors decreased 3-NT levels and associated pathology. 3-NT determination allowed for better elucidation of the role of iNOS in RA and OA disease pathology and provided proof of pharmacology for NOS inhibitors in animal models of RA and OA.

Preanalytical approaches to improve recovery of amyloid-ß peptides from CSF as measured by immunological or mass spectrometry-based assays.

BACKGROUND: Amyloid-ß 1-42 (Aß1-42) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer's disease (AD). Reduced levels of Aß1-42 are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aß recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aß peptides in stored samples. METHODS: CSF from healthy control subjects and patients with AD was aliquoted into polypropylene tubes at volumes of 0.1 ml and 0.5 ml. CSF Aß1-42 concentrations were initially measured by immunoassay; subsequent determinations of CSF Aß1-42, Aß1-40, Aß1-38, Aß1-37, and Aß1-34 concentrations were made with an absolute quantitative mass spectrometry assay. In a second study, CSF from healthy control subjects and patients with dementia was denatured with guanidine hydrochloride (GuHCl) at different stages of the CSF collection and aliquoting process and then measured with the mass spectrometry assay. RESULTS: Two distinct immunoassays demonstrated that CSF Aß1-42 concentrations measured from 0.5-ml aliquots were higher than those from 0.1-ml aliquots. Tween-20 surfactant supplementation increased Aß1-42 recovery but did not effectively resolve measured concentration differences associated with aliquot size. A CSF Aß peptide mass spectrometry assay confirmed that Aß peptide recovery was linked to sample volume. Unlike the immunoassay experiments, measured differences were consistently eliminated when aliquots were denatured in the original sample tube. Recovery from a panel of low-retention polypropylene tubes was assessed, and 1.5-ml Eppendorf LoBind® tubes were determined to be the least absorptive for Aß1-42. A comparison of CSF collection and processing methods suggested that Aß peptide recovery was improved by denaturing CSF earlier in the collection/aliquoting process and that the Aß1-42/Aß1-40 ratio was a useful method to reduce variability. CONCLUSIONS: Analyte loss due to nonspecific sample tube adsorption is a significant preanalytical factor that can compromise the accuracy of CSF Aß1-42 measurements. Sample denaturation during aliquoting increases recovery of Aß peptides and improves measurement accuracy. The Aß1-42/Aß1-40 ratio can overcome some of the quantitative variability precipitated by preanalytical factors affecting recovery.

Clinical validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide: A biomarker of matrix metalloproteinase activity and osteoarthritis in human urine.

The degradation of type II collagen has been associated with the pathology of osteoarthritis (OA). Matrix metalloproteinases (MMPs) are enzymes that are responsible for catalyzing the degradation of collagen and, therefore, are pursued as potential targets for the treatment of OA. Collagen-derived peptides identified as a reflection of in vivo MMP activity have been investigated as target biomarkers of MMP activity as well as potential biomarkers of OA disease state and/or progression. An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay developed for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues resulting from MMP-13-catalyzed degradation, was validated for clinical use. Validation experiments were designed with attention to specific challenges related to quantification of endogenous analytes. The validated method is sensitive, selective, accurate (<15% relative error) and precise (<15% coefficient of variation) over a linear range of 0.156-7.50 ng/ml. Sample stability and inter- and intrasubject variability were evaluated in the urine of normal and OA populations. The method was applied to analyze human urine samples from clinical studies investigating the utility of uTIINE as a potential biomarker for OA.