Neopterin is synthesized by macrophages upon stimulation with gamma-interferon, and high neopterin production is associated with cellular immune activation and increased production of reactive oxygen species (oxidant stress), but the clinical utility of urine neopterin levels in patients with heart failure (HF) has not been explored. Fifty-three ambulatory patients with chronic systolic HF (left ventricular [LV] ejection fraction ≤40%) underwent comprehensive echocardiographic evaluation and cardiopulmonary exercise testing. Urine neopterin levels were quantified by liquid chromatography with tandem mass spectrometric analyses and corrected to urine creatinine (Cr) levels. In our study cohort, median urine neopterin level was 60 μmol/mol Cr (interquartile range 40 to 86). There were modest correlations between urine neopterin levels and abnormalities in cardiac structure and function by echocardiography: LV ejection fraction (r = −0.33, p = 0.017), indexed LV end-diastolic volume (r = 0.31, p = 0.029), indexed LV end-systolic volume (r = 0.32, p = 0.024), and E/septal Ea (r = 0.28, p = 0.041). Although there was no significant correlation between urine neopterin and maximal oxygen uptake (peak V O2 : r = −0.25, p = 0.07), there was a modest correlation between urine neopterin and maximal ventilation/carbon dioxide production ratio (V E /V CO2 max: r = 0.38, p = 0.005). In conclusion, increase in urine neopterin levels tracks with disease severity in patients with chronic systolic HF.
Neopterin is a pteridine that is produced as a catabolic product of guanosine triphosphate metabolism. It is synthesized and released by activated monocytes or macrophages upon stimulation with gamma-interferon. Increased neopterin levels indicate endogenous formation of gamma-interferon produced by activated T cells (Th1 or cytotoxic T cells) and suggest cell-mediated immune activation. Neopterin is produced together with tetrahydrobiopterin, which is a cofactor for oxidative cleavage of ether lipids and formation of nitric oxide from arginine. High neopterin level is associated with increased production of reactive oxygen species, suggesting oxidative stress elicited by the immune system. Previous studies have reported that serum neopterin levels may be elevated in patients with coronary and peripheral artery diseases, correlate with the extent of disease and complex (vulnerable) plaques, and also predict major adverse cardiovascular events in patients with chronic coronary artery disease, acute coronary syndromes, or severe peripheral artery disease. In addition, it has been shown that patients with congestive heart failure (HF) had higher blood neopterin levels, which correlated with the severity of HF with advanced signs and symptoms, and independently predicted future cardiac events for patients with chronic HF. For acute decompensated HF, higher serum neopterin levels predict increased risk of developing renal dysfunction. All these studies utilized circulating neopterin, although neopterin has long been isolated in the urine, and the value and reproducibility of urine neopterin level in patients with HF has not been determined. Utilizing mass spectrometry techniques, this study is to explore the application of urine neopterin in patients with chronic systolic HF and its relation with severity of HF.
Methods
Fifty-three consecutively consented ambulatory patients with stable systolic HF were prospectively enrolled to participate in a blood draw, urine collection, echocardiography, and cardiopulmonary exercise testing. To participate, patents had to have a clinical diagnosis of chronic (>6 months’ duration) HF with left ventricular (LV) ejection fraction ≤40% by echocardiography in euvolemic state at the time of enrollment. We excluded subjects with a major cardiovascular event (myocardial infarction, unstable angina, stroke, transient ischemic attack, and pulmonary embolism) within 30 days of enrollment. Other exclusion criteria including a history of significant chronic obstructive pulmonary disease, major surgery, hospitalization, or emergency room visits for HF exacerbation or use of inotropic agents within the month preceding enrollment. This study was approved by the Cleveland Clinic Institutional Review Board, and all subjects provided written informed consent.
A comprehensive 2-dimensional echocardiography was performed with a commercially available system (Vingmed, System Seven, General Electric, Piscataway, New Jersey) by a single American Society of Echocardiography registered research sonographer. Images were acquired by a dedicated research sonographer in the left lateral decubitus position with a phased-array transducer in the standard parasternal and apical views. Standard 2-dimensional and Doppler data, triggered to the QRS complex, were digitally stored in a cine-loop format and analyzed in a blinded fashion from assay results. Subjects then underwent a symptom-limited cardiopulmonary stress testing using the modified Naughton protocol, and results were recorded on a Vmax Encore Metabolic Cart (CareFusion, San Diego, California), and peak oxygen consumption and ventilator efficiency (assessed by the ratio of maximal ventilation/carbon dioxide production) were measured according to the clinical standards.
Urine samples were collected at spot collection at midstream and in morning fasting condition and centrifuged at 1,500× g for 10 minutes at 4°C. Samples were then stored in aliquots at −80°C and thawed on ice before analysis. Because neopterin is slightly sensitive to direct sunlight irradiation, samples were protected from light during transport and storage. Urine aliquots (200 μl) are mixed with 200 ng/ml rhamnopterin (internal standard) and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected, and an aliquot is injected onto a high-performance liquid chromatography column, and neopterin levels were quantified by mass spectrometry using Waters Quattro Ultima (Micromass, Milford, Massachusetts) connected to a Waters Alliance 2690 HPLC system. Urine pterins were separated on a 2 × 150 mm C18, ODS (2), 5 μm, 150 Å column (Phenomenex, Torrance, California) and resolved using a discontinuous gradient with 0.2% formic acid (solvent A) and 0.2% formic acid in methanol (solvent B). The gradient used was as follows: the column was first equilibrated with 100% solvent A for 2 minutes after the injection; a linear gradient was then run to 50% solvent B over the next 6 minutes and held for 4 minutes. Then the solvent was changed to 100% solvent A in a linear fashion over 0.5 minutes, held at 100% solvent A for 7.5 minutes. Flow rate was kept at 200 μl/min. Analyses were performed online using liquid chromatography tandem mass spectrometry in the positive ion mode with multiple reaction monitoring using unique parent→daughter ion transition (254→206) and retention time for neopterin. Cone potential and collision energy are optimized for neopterin, and the standard curve was generated with rhamnopterin as internal standard. Intra-assay and inter-assay coefficients of variance were <10%. Urine creatinine (Cr, for correction of urine excretion) and B-type natriuretic peptide levels were measured by the Architect ci8200 platform (Abbott Laboratories, Abbott Park, Illinois).
Continuous variables are summarized as mean ± SD if normally distributed or as median (interquartile range) if non-normally distributed. Normality was assessed by the Shapiro-Wilk W test. Differences in normally distributed continuous variables across clinical categories were assessed using the Student t test, whereas differences in non-normally distributed variables were assessed using the Wilcoxon rank-sum test. Univariate Spearman’s correlation analysis was used to determine the correlation between urine neopterin levels and echocardiographic and cardiopulmonary testing indices. All p values reported were from 2-sided tests, and p value of <0.05 was considered statistically significant. Statistical analyses were performed using JMP 9.0.0 (SAS Institute, Cary, North Carolina).
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