Speciation and quantification of thiols by reversed-phase chromatography coupled with on-line chemical vapor generation and atomic fluorescence spectrometric detection: method validation and preliminary application for glutathione measurements in human whole blood.

BACKGROUND: We developed a sensitive, specific method for the low-molecular-mass thiols cysteine, cysteinylglycine, glutathione, and homocysteine and validated the method for measurement of glutathione in blood. METHODS: The technique was based on reversed-phase chromatography (RPC) coupled on line with cold vapor generation atomic fluorescence spectrometry (CVGAFS). Thiols were derivatized before introduction on the column by use of a p-hydroxymercuribenzoate (PHMB) mercurial probe and separated as thiol-PHMB complexes on a Vydac C4 column. Postcolumn on-line reaction of derivatized thiols with bromine allowed rapid conversion of the thiol-PHMB complexes to inorganic mercury with recovery of 100 (2)% of the sample. HgII was selectively detected by atomic fluorescence spectrometry in an Ar/H2 miniaturized flame after sodium borohydride reduction to Hg0. RESULTS: The relationship between thiol-PHMB complex concentration and peak area (CVGAFS signal) was linear over the concentration range 0.01-1400 micromol/L (injected). The detection limits were 1, 1, 0.6, and 0.8 nmol/L for cysteine, cysteinylglycine, homocysteine, and glutathione in the injected sample, respectively. The CVs for thiols were 1.5%-2.2% for calibrator solutions and 2.1% and 3.0% for real samples. The RPC-CVGAFS method allowed speciation of glutathione (reduced and oxidized) in human whole blood from healthy donors and from the coronary sinus of patients with idiopathic dilated cardiomyopathy during and after chronotropic stress. CONCLUSION: The RPC-CVGAFS method could be used to measure reduced and oxidized glutathione in human whole blood as disease biomarkers.

Respiratory changes in transvalvular flow velocities versus two-dimensional echocardiographic findings in the diagnosis of cardiac tamponade.

BACKGROUND: The purpose of this study was to compare the sensitivity, specificity and positive predictive value of the respiratory changes in the transvalvular flow velocities to those of right atrial collapse and right ventricular collapse in the diagnosis of cardiac tamponade. METHODS: Standard two-dimensional and Doppler echocardiography were performed with respiratory monitoring in 56 consecutive patients with mild to severe pericardial effusion. Sixteen patients met the clinical criteria for cardiac tamponade and underwent pericardiocentesis or surgical drainage. Forty patients were found to have no tamponade and were followed up for at least 2 weeks and none of them showed clinical worsening. RESULTS: The sensitivity, specificity and predictive value were, respectively, 77, 80 and 62% for an inspiratory decrease > 22% in the peak velocity of the early mitral flow; 75, 89 and 73% for an inspiratory reduction > 20% in the peak velocity of the aortic flow; 50, 69 and 36% for an inspiratory increase > 30% in the peak velocity of the early tricuspid flow; 87, 85 and 64% for an inspiratory increase > 25% in the peak velocity of the pulmonary flow. Right atrial collapse and right ventricular collapse had a sensitivity of 100 and 75%, a specificity of 33 and 85%, and a predictive value of 37 and 66%, respectively. CONCLUSIONS: In the diagnosis of cardiac tamponade: 1) right atrial collapse is the most sensitive sign but lacks any specificity; 2) except for the tricuspid valve, the respiratory variations in the transvalvular flow velocities have a reliability and a predictive value comparable with those of right ventricular collapse; 3) the predictive value is not very high, indicating that at both techniques false positive results are not negligible.