Serum and urine lipidomic profiles identify biomarkers diagnostic for seropositive and seronegative rheumatoid arthritis.

Objective: Seronegative rheumatoid arthritis (RA) is defined as RA without circulating autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies; thus, early diagnosis of seronegative RA can be challenging. Here, we aimed to identify diagnostic biomarkers for seronegative RA by performing lipidomic analyses of sera and urine samples from patients with RA. Methods: We performed untargeted lipidomic analysis of sera and urine samples from 111 RA patients, 45 osteoarthritis (OA) patients, and 25 healthy controls (HC). These samples were divided into a discovery cohort (n = 97) and a validation cohort (n = 84). Serum samples from 20 patients with systemic lupus erythematosus (SLE) were also used for validation. Results: The serum lipidome profile of RA was distinguishable from that of OA and HC. We identified a panel of ten serum lipids and three urine lipids in the discovery cohort that showed the most significant differences. These were deemed potential lipid biomarker candidates for RA. The serum lipid panel was tested using a validation cohort; the results revealed an accuracy of 79%, a sensitivity of 71%, and a specificity of 86%. Both seropositive and seronegative RA patients were differentiated from patients with OA, SLE, and HC. Three urinary lipids showing differential expression between RA from HC were identified with an accuracy of 84%, but they failed to differentiate RA from OA. There were five lipid pathways that differed between seronegative and seropositive RA. Conclusion: Here, we identified a panel of ten serum lipids as potential biomarkers that can differentiate RA from OA and SLE, regardless of seropositivity. In addition, three urinary lipids had diagnostic utility for differentiating RA from HC.

Immunodetection of urinary C-terminal telopeptide fragment of type II collagen: An osteoarthritis biomarker analysis.

The urinary C-terminal telopeptide fragment of type II collagen (uCTX-II) has been reported as the efficient blood-based biomarker for osteoarthritis, which affects knees, hands, spine, and hips. This study reports a sensing strategy with antibody-conjugated gold nanoparticles (GNP) on an interdigitated electrode (IDE) to determine uCTX-II. The GNP-antibody complex was chemically immobilized on the IDE surface through the amine linker. uCTX-II was determined by monitoring the alteration in current upon interacting the GNP-complexed antibody. This strategy was improved the detection by attracting higher uCTX-II molecules, and the detection limit falls in the range of 10-100 pM with an acceptable regression value [y = 0.6254x - 0.4073, R² = 0.9787]. The sensitivity of the detection was recognized at 10 pM. Additionally, upon increasing the uCTX-II concentration, the current changes were increased in a linear fashion. Control detection with nonimmune antibody and control protein do not increase the current level, confirming the specific detection of uCTX-II. This method of detection helps in diagnosing osteoarthritis and its follow-up treatment.

Associations of urinary levels of phenols and parabens with osteoarthritis among US adults in NHANES 2005-2014.

Phenols and parabens are two major classes of endocrine-disrupting compounds (EDCs) that may be related to multiple human diseases. However, there has been no studies examining the association between phenols as well as parabens and osteoarthritis (OA). We assessed the link between urinary concentrations of triclosan (TCS), benzophenone-3 (BP-3), bisphenol A (BPA), and parabens with OA based on the data collected from National Health and Nutrition Examination Survey in multivariable logistic regression models. Among all the 7114 participants included, the weighted percentage of OA was 12.11% (n = 807). Compared with participants at tertile 1, those at tertile 2 of urinary BP-3, and tertile 3 of urinary BP-3 were more likely to show increased OA prevalence in a fully adjusted model, with odd ratio (OR) as 1.34 [95% confidence interval (CI): 1.01-1.78], 1.55 (95 CI%: 1.17-2.06), and 1.66 (95 CI%: 1.23-2.24), respectively. In subgroup analyses stratified by potential confounders, various subgroups remained to show statistically significant positive association between urinary BP-3 and OA prevalence. Otherwise, we observed no statistically significant associations between urinary TCS, BPA or parabens with OA. In conclusion, this serves as the first study in which we found that the urinary concentration of BP-3 was positively correlated to prevalence of OA among the US population.

Blood and urinary collagen markers in osteoarthritis: markers of tissue turnover and disease activity.

Introduction: The need for diagnostic markers in osteoarthritis (OA) is acute and immediate, as sensitive and precise tools that monitor disease activity and treatment response are lacking. Collagens - types I, II, and III - are the skeleton of the extracellular matrix of joint tissues. Joint collagens are generally turned over at a low rate, but the balance between formation and degradation is disturbed, leading to the loss of, for example, cartilage.Areas covered: We discuss the markers reflecting collagen turnover and provide examples of how they have been applied in OA research, as well as how we believe these should be used in the future. We have searched PubMed for full-text articles written in English using different combinations of the following terms: OA, biomarker, and collagen. The result is a narrative review that gives examples from the literature.Expert opinion: Collagen markers show promise, as they are direct measures of tissue balance. Until now, collagen markers have mainly been tested in observational cohorts, which may provide insights into the association between the candidate marker and clinical variables; however, these do not advance the development of qualified markers that can be used for drug development or in clinical practice.

Advances in Molecular biomarker for early diagnosis of Osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative joint disease. The pathogenesis is poorly understood. What is known is that OA is characterized by imbalance in anabolic and catabolic gene expression in articular chondrocytes. This results in bone on bone articulations resulting in impaired mobility and joint pain. Although the cause of OA is unknown, comorbidities include: aging, obesity, and mechanical stress. Currently the only diagnostic modalities are radiology and physical examination, and early detection is rare. Biomarkers are quantifiable substances, and their presence can be suggestive of a certain phenomenon or disease. Biomarkers are popular for early diagnosis for pathological conditions in the fields of oncology, cardiology, and endocrinology. This review has systematically reviewed the literature about biomarkers in the field of OA, specifically protein, miRNA, and metabolic biomarkers found in the blood, urine, and synovial fluid.

Establishment of reference intervals for osteoarthritis-related soluble biomarkers: the FNIH/OARSI OA Biomarkers Consortium.

OBJECTIVE: To establish reference intervals for osteoarthritis (OA)-related biomarkers used in the Foundation for the National Institutes of Health (FNIH) OA Biomarkers Consortium Project. METHODS: A total of 129 'multijoint controls' were selected from 2722 African-American and Caucasian men and women in the Johnston County Osteoarthritis Project. The majority (79%) of those eligible (with biospecimens and baseline data) also had one or more follow-up evaluations 5-15 years later. Multijoint controls were selected to be free of radiographic hand, hip, knee and lumbar spine osteoarthritis (OA), to have no knee or hip symptoms, and minimal hand and spine symptoms at all available time points. Eighteen biomarkers were evaluated in serum (s) and/or urine (u) by ELISA. Reference intervals and partitioning by gender and race were performed with EP Evaluator software. RESULTS: Controls were 64% women, 33% African-Americans, mean age 59 years and mean body mass index 29 kg/m2. Three biomarkers were associated with age: sHyaluronan (positively), sN-terminal propeptide of collagen IIA (positively) and sCol2-3/4 C-terminal cleavage product of types I and II collagen (negatively). Exploratory analyses suggested that separate reference intervals may be warranted on the basis of gender for uC-terminal cross-linked telopeptide of type II collagen (uCTXII), sMatrix metalloproteinase-3, uNitrated type II collagen degradation fragment (uCol2-1 NO2) and sHyaluronan, and on the basis of race for uCTXII, sCartilage oligomeric matrix protein, sC-terminal cross-linked telopeptide of type I collagen and uCol2-1 NO2. CONCLUSIONS: To our knowledge, this represents the best and most stringent control group ever assayed for OA-related biomarkers. These well-phenotyped controls, representing a similar age demographic to that of the OA Initiative-FNIH main study sample, provide a context for interpretation of OA subject biomarker data. The freely available data set also provides a reference for future human studies.

Advances in urinary protein biomarkers for urogenital and non-urogenital pathologies.

The discovery of protein biomarkers that reflect the biological state of the body is of vital importance to disease management. Urine is an ideal source of biomarkers that provides a non-invasive approach to diagnosis, prognosis and prediction of diseases. Consequently, the study of the human urinary proteome has increased dramatically over the last 10 years, with many studies being published. This review focuses on urinary protein biomarkers that have shown potential, in initial studies, for diseases affecting the urogenital tract, specifically chronic kidney disease and prostate cancer, as well as other non-urogenital pathologies such as breast cancer, diabetes, atherosclerosis and osteoarthritis. PubMed was searched for peer-reviewed literature on the subject, published in the last 10 years. The keywords used were "urine, biomarker, protein, and/or prostate cancer/breast cancer/chronic kidney disease/diabetes/atherosclerosis/osteoarthritis". Original studies on the subject, as well as a small number of reviews, were analysed including the strengths and weaknesses, and we summarized the performance of biomarkers that demonstrated potential. One of the biggest challenges found is that biomarkers are often shared by several pathologies so are not specific to one disease. Therefore, the trend is shifting towards implementing a panel of biomarkers, which may increase specificity. Although there have been many advances in urinary proteomics, these have not resulted in similar advancements in clinical practice due to high costs and the lack of large data sets. In order to translate these potential biomarkers to clinical practice, vigorous validation is needed, with input from industry or large collaborative studies.

Treatment with recombinant lubricin attenuates osteoarthritis by positive feedback loop between articular cartilage and subchondral bone in ovariectomized rats.

Osteoarthritis (OA) is a most commonly multifactorial degenerative joint disease along with the aging population, particularly in postmenopausal women. During the onset of OA, articular cartilage and subchondral bone act in concert as a functional unit. This present study is to investigate the effects of early or late treatment with recombinant lubricin on the onset of osteoarthritis (OA) in ovariectomized (OVX) rats. We found that both early and late recombinant lubricin treatments attenuated the onset of OA by positive feedback loop between articular cartilage and subchondral bone, although late treatment contributed to a lesser effect compared with early treatment. Specifically, treatment with recombinant lubricin protected articular cartilage from degeneration, demonstrated by lower proteoglycan loss, lower OARSI scores, less calcification cartilage zone and reduced immunostaining for collagen X (Col X) and matrix metalloproteinase (MMP-13) but increased the expression of lubricin, in comparison with vehicle-treated OVX rat group. Further, chondroprotective effects of lubricin normalized bone remodeling in subchondral bone underneath. It's suggested that treatment with recombinant lubricin inhibited the elevation of TRAP and Osterix positive cells in OVX rats and led to the normalization of subchondral bone microarchitectures with the suppression of subsidence of bone volume ratio (BV/TV) and trabecular thickness (Tb.Th) and the increase of trabecular separation (Tb.Sp) in vehicle-treated OVX rats. What's more, the normalization of subchondral bone in turn attenuated the articular cartilage erosion by inhibiting vascular invasion from subchondral bone to calcified cartilage zone, exemplified by inhibiting the elevation of CD31 positive cells in calcified cartilage and angiography in subchondral bone. Together, these results shed light that both early and late recombinant lubricin treatments attenuate the onset of OA by balancing the interplay between articular cartilage and subchondral bone in OVX rats, while also providing a further rationale for its therapeutic targeting to postmenopausal OA and suggesting that treatment timing is a pivotal factor for better effect acquisition.

[THE ANALYSIS OF INDICATORS OF MINERAL METABOLISM IN PATIENTS WITH DEGENERATIVE DYSTROPHIC AFFECTIONS OF JOINTS].

The analysis of indicators of mineral metabolism in patients with degenerative dystrophic affections of joints demonstrated that under development of osteoarthrosis process the alteration of indicators of concentration of electrolytes in blood serum, urine and synovial fluid occurs. The stage II of process is characterized by maximal alterations of indicators. The indicator of relationship between concentration of phosphate-ion and index of phosphatases of blood serum turned out the significant coefficient of correlation.

Urinary proteome profile predictive of disease activity in rheumatoid arthritis.

Current serum biomarkers for rheumatoid arthritis (RA) are not highly sensitive or specific to changes of disease activities. Thus, other complementary biomarkers have been needed to improve assessment of RA activities. In many diseases, urine has been studied as a window to provide complementary information to serum measures. Here, we conducted quantitative urinary proteome profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified 134 differentially expressed proteins (DEPs) between RA and osteoarthritis (OA) urine samples. By integrating the DEPs with gene expression profiles in joints and mononuclear cells, we initially selected 12 biomarker candidates related to joint pathology and then tested their altered expression in independent RA and OA samples using enzyme-linked immunosorbent assay. Of the initial candidates, we selected four DEPs as final candidates that were abundant in RA patients and consistent with those observed in LC-MS/MS analysis. Among them, we further focused on urinary soluble CD14 (sCD14) and examined its diagnostic value and association with disease activity. Urinary sCD14 had a diagnostic value comparable to conventional serum measures and an even higher predictive power for disease activity when combined with serum C-reactive protein. Thus, our urinary proteome provides a diagnostic window complementary to current serum parameters for the disease activity of RA.

Performance of different screening methods for the determination of urinary glycosaminoclycans.

BACKGROUND: The study aim was to compare the performance of three different methods used for determining urinary glycosaminoglycans (GAG) levels in spot and 24-h urine samples. METHODS: Performance characteristics were studied for cetylpyridinium chloride (CPC), and manual and automated dimethylmethylene blue (DMB) methods. RESULTS: For automated DMB method, within-run precisions were 9.10% and 1.98%, and between-day precisions were 13.0% and 5.81% in low- and high-urine pools, respectively. The method was linear up to 100 mg/L of GAG concentration. The detection limit of the method was 0.71 mg/L. Mean recovery was 95.7%. CONCLUSIONS: The automated DMB method was found to give better performance characteristics than cetylpyridinium chloride (CPC) and manual DMB methods. It is a fast, cheap, simple and reliable method and can be applied in many diseases in which GAG is used as a screening test.

Development of a novel immunoassay for the measurement of type II collagen neoepitope generated by collagenase cleavage.

BACKGROUND: To evaluate cartilage degeneration in arthritis, we developed a novel enzyme-linked immunosorbent assay (ELISA) with the capacity to determine urinary concentrations of type II collagen neoepitope (CIINE) generated by collagenase cleavage. METHODS: Two monoclonal antibodies, 20A10 and 6G4, were generated. Of these antibodies, 20A10 recognized CIINE regardless of hydroxylation of Pro77¹, and 6G4 recognized the type II collagen-specific region adjacent to the neoepitope. A sandwich ELISA was constructed using these antibodies. RESULTS: The ELISA positively determined CIINE concentrations from human and dog urine samples, and from tissue culture supernatant of rat and bovine cartilage. Validation with human urine samples revealed that the ELISA had a detection limit of 100 pmol/l, with intra- and inter-assay coefficients of less than 15%. Recovery of extraneously added CIINE peptide to human urine samples was 83.1-104%. Measurement with the ELISA demonstrated that urine samples from OA patients contained CIINE at significantly higher concentrations compared with those from healthy controls (P<0.01). CONCLUSIONS: The ELISA can determine the CIINE concentration in human urine sensitively and accurately. This assay may also be useful to determine the concentration of CIINE of various animal samples.

Urinary glycosaminoglycans in horse osteoarthritis. Effects of chondroitin sulfate and glucosamine.

Our objectives were to characterize the urinary excretion of glycosaminoglycans (GAGs) in horse osteoarthritis, and to investigate the effects of chondroitin sulfate (CS) and glucosamine (GlcN) upon the disease. Urinary GAGs were measured in 47 athletic horses, 20 healthy and 27 with osteoarthritis. The effects of CS and GlcN were investigated in mild osteoarthritis. In comparison to normal, urinary GAGs were increased in osteoarthritis, including mild osteoarthritis affecting only one joint. Treatment with CS+GlcN led to a long lasting increase in the urinary CS and keratan sulfate (KS), and significant improvement in flexion test of tarsocrural and metacarpophalangeal joints was observed. In conclusion, urinary CS and KS seems to reflect the turnover rates of cartilage matrix proteoglycans, and the measurement of these compounds could provide objective means of evaluating and monitoring joint diseases.

Biomarkers of cartilage turnover. Part 1: Markers of collagen degradation and synthesis.

Type II collagen is a major component of articular cartilage and its breakdown is a key feature of osteoarthritis. Products of cartilage collagen metabolism can be detected in the blood, synovial fluid and urine. Several biomarker assays have been developed which can be used to measure the synthesis and degradation of collagen, and therefore provide information regarding cartilage turnover. This is the first part of a two-part review and describes the need for accurate, reliable information regarding collagen turnover, the processes by which the biomarker epitopes are generated, their application to the study of both healthy and diseased cartilage and the results of currently published studies, with particular reference to the veterinary species. The second part of the review considers the non-collagenous biomarkers of cartilage matrix turnover.

Design and validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide in human urine: application as a biomarker of osteoarthritis.

Biomarkers play an increasingly important role for drug efficacy and safety evaluation in all stages of drug development. It is especially important to develop and validate sensitive and selective biomarkers for diseases where the onset of the disease is very slow and/or the disease progression is hard to follow, i.e., osteoarthritis (OA). The degradation of Type II collagen has been associated with the disease state of OA. Matrix metalloproteinases (MMPs) are enzymes that catalyze the degradation of collagen and therefore pursued as potential targets for the treatment of OA. Peptide biomarkers of MMP activity related to type II collagen degradation were identified and the presence of these peptides in MMP digests of human articular cartilage (HAC) explants and human urine were confirmed. An immunoaffinity LC/MS/MS assay for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues was developed and clinically validated. The assay has subsequently been applied to analyze human urine samples from clinical studies. We have shown that the assay is able to differentiate between symptomatic OA and normal subjects, indicating that uTIINE can be used as potential biomarker for OA. This chapter discusses the assay procedure and provides information on the validation experiments used to evaluate the accuracy, precision, and selectivity data with attention to the specific challenges related to the quantification of endogenous protein/peptide biomarker analytes. The generalized approach can be used as a follow-up to studies whereby proteomics-based urinary biomarkers are identified and an assay needs to be developed. Considerations for the validation of such an assay are described.

Thrombin-cleaved osteopontin is increased in urine of patients with rheumatoid arthritis.

OBJECTIVE: To measure concentrations of the thrombin-cleaved isoform of osteopontin (OPN) in urine and plasma of patients with rheumatoid arthritis (RA), and to assess whether levels of thrombin-cleaved OPN are associated with measures of RA. METHODS: Subjects comprised 70 patients with RA, 20 patients with osteoarthritis (OA), and 46 healthy controls. RA disease activity was evaluated by tender joint count, swollen joint count, patient's global assessment of disease activity, erythrocyte sedimentation rate (ESR), and levels of C-reactive protein (CRP), matrix metalloproteinase-3 (MMP-3), and rheumatoid factor (RF), as well as 28-joint count Disease Activity Score (DAS28). OPN levels in plasma and urine were measured by ELISA. RESULTS: Median levels of thrombin-cleaved OPN in urine (U-half) were significantly higher in RA patients (143.5 pmol/mmol Cr) than in healthy controls (67.9 pmol/mmol Cr) or OA patients (69.8 pmol/mmol Cr). Thrombin-cleaved OPN was not detected in plasma. U-half levels correlated significantly with levels of CRP (r = 0.26, p = 0.03), ESR (r = 0.26, p = 0.03), and RF (r = 0.28, p = 0.03). Median U-half levels were significantly higher in patients with stage III (249.9 pmol/mmol Cr) and IV (251.6 pmol/mmol Cr) disease than in patients with stage I (98.6 pmol/mmol Cr) disease. CONCLUSION: Our results suggest that urine levels of the thrombin-cleaved isoform of OPN may reflect the severity of active inflammatory arthritis in patients with RA.

A longitudinal analysis of urinary biochemical markers and bone mineral density in STR/Ort mice as a model of spontaneous osteoarthritis.

OBJECTIVE: To investigate the longitudinal changes both in the urinary concentrations of biochemical markers and in bone mineral density (BMD) during disease progression in the STR/Ort mouse model of osteoarthritis (OA). METHODS: Male STR/Ort mice were studied, with CBA mice used as nonarthritic controls. Radiographic evaluation and grading of the knee and measurements of urinary C-terminal crosslinking telopeptide of type II collagen (CTX-II), pyridinoline (Pyr), and deoxypyridinoline were performed between 8 weeks and 40 weeks of age. The BMD of the femoral shaft was measured from 20 weeks to 40 weeks of age and adjusted for body weight. Histologic evaluation and grading were performed at 40 weeks of age. STR/Ort mice were divided into 2 subgroups (STR OA and STR non-OA) based on histologic grading. RESULTS: No significant differences between STR/Ort and CBA mice were observed for any biochemical marker or BMD at any time point. Urinary CTX-II levels and BMD in the STR OA subgroup were higher than those in the STR non-OA subgroup before radiographic changes of OA were apparent. Higher urinary Pyr levels in the STR OA subgroup were observed at the advanced stage of OA. CONCLUSION: Urinary CTX-II could be a useful marker in the early diagnosis and predicting the progression of OA, and urinary Pyr may be a potential marker to assess the severity of OA at an advanced stage. An increase in BMD prior to the establishment of radiographic OA may be related to the induction of OA.

Detection of cartilage oligomeric matrix protein using a quartz crystal microbalance.

Current methods for diagnosing early stage osteoarthritis (OA) based on the magnetic resonance imaging and enzyme-linked immunosorbent assay methods are specific, but require specialized laboratory facilities and highly trained personal to obtain a definitive result. In this work, a user friendly and non-invasive quartz crystal microbalance (QCM) immunosensor method has been developed to detect Cartilage Oligomeric Matrix Protein (COMP) for early stage OA diagnosis. This QCM immunosensor was fabricated to immobilize COMP antibodies utilizing the self-assembled monolayer technique. The surface properties of the immunosensor were characterized by its FTIR and electrochemical impedance spectra (EIS). The feasibility study was based on urine samples obtained from 41 volunteers. Experiments were carried out in a flow system and the reproducibility of the electrodes was evaluated by the impedance measured by EIS. Its potential dynamically monitored the immunoreaction processes and could increase the efficiency and sensitivity of COMP detection in laboratory-cultured preparations and clinical samples. The frequency responses of the QCM immunosensor changed from 6 kHz when testing 50 ng/mL COMP concentration. The linear regression equation of frequency shift and COMP concentration was determined as: y=0.0872 x+1.2138 (R2=0.9957). The COMP in urine was also determined by both QCM and EIS for comparison. A highly sensitive, user friendly and cost effective analytical method for the early stage OA diagnosis has thus been successfully developed.

Identification of progressors in osteoarthritis by combining biochemical and MRI-based markers.

INTRODUCTION: At present, no disease-modifying osteoarthritis drugs (DMOADS) are approved by the FDA (US Food and Drug Administration); possibly partly due to inadequate trial design since efficacy demonstration requires disease progression in the placebo group. We investigated whether combinations of biochemical and magnetic resonance imaging (MRI)-based markers provided effective diagnostic and prognostic tools for identifying subjects with high risk of progression. Specifically, we investigated aggregate cartilage longevity markers combining markers of breakdown, quantity, and quality. METHODS: The study included healthy individuals and subjects with radiographic osteoarthritis. In total, 159 subjects (48% female, age 56.0 +/- 15.9 years, body mass index 26.1 +/- 4.2 kg/m2) were recruited. At baseline and after 21 months, biochemical (urinary collagen type II C-telopeptide fragment, CTX-II) and MRI-based markers were quantified. MRI markers included cartilage volume, thickness, area, roughness, homogeneity, and curvature in the medial tibio-femoral compartment. Joint space width was measured from radiographs and at 21 months to assess progression of joint damage. RESULTS: Cartilage roughness had the highest diagnostic accuracy quantified as the area under the receiver-operator characteristics curve (AUC) of 0.80 (95% confidence interval: 0.69 to 0.91) among the individual markers (higher than all others, P < 0.05) to distinguish subjects with radiographic osteoarthritis from healthy controls. Diagnostically, cartilage longevity scored AUC 0.84 (0.77 to 0.92, higher than roughness: P = 0.03). For prediction of longitudinal radiographic progression based on baseline marker values, the individual prognostic marker with highest AUC was homogeneity at 0.71 (0.56 to 0.81). Prognostically, cartilage longevity scored AUC 0.77 (0.62 to 0.90, borderline higher than homogeneity: P = 0.12). When comparing patients in the highest quartile for the longevity score to lowest quartile, the odds ratio of progression was 20.0 (95% confidence interval: 6.4 to 62.1). CONCLUSIONS: Combination of biochemical and MRI-based biomarkers improved diagnosis and prognosis of knee osteoarthritis and may be useful to select high-risk patients for inclusion in DMOAD clinical trials.

Amino acid profiling in urine by capillary zone electrophoresis - mass spectrometry.

Analysis of amino acid profiles in urine and plasma is an essential part of modern clinical diagnostic routine. Here we present an approach for the analysis of amino acids in urine by capillary electrophoresis/time-of-flight (TOF) mass spectrometry. At first a method combining improved separation, high dynamic range, and high sensitivity is presented. Detection limits in the mid nM-range are achieved through the use of pH-mediated stacking injection in combination with modern TOF detection technology. The method can be easily applied to detect differences in the amino acid profile in urine in a clinical context. Moreover, beside amino acids low molecular weight amines, peptides and related metabolites can be profiled. As a proof of concept, urine samples from patients suffering from osteoarthritis have been analyzed. Finally, the introduction of multivariate data analysis in the work flow was evaluated on spiked urine samples and real clinical material.