Development of an immunoassay for the derived-peptide of chromogranin A, vasostatin-I (1-76): assessment of severity in patients with sepsis.

CONTEXT: Proteolytic fragments of chromogranin A (CgA) including the CgA 1-76 fragment (called vasostatin-I [VS-I]) could be a useful biomarker of sepsis, but there is no available immunoassay. METHODS: A sandwich ELISA for VS-I was developed, and plasma VS-I was measured in 30 healthy controls and 60 critically ill patients with sepsis. RESULTS: The ELISA showed intra- and inter-assay coefficients of variations (CVs) below 4 and 9%. Plasma VS-I was significantly increased compared with controls in patients with sepsis, severe sepsis, and sepsis shock (p < 0.0001). Receiver operating curve (ROC) analyses indicated that plasma VS-I was more sensitive and specific than plasma CgA to diagnose sepsis and to assess its severity. CONCLUSIONS: The measurements of plasma VS-I with this new ELISA may be useful for the clinical investigation of patients with sepsis.

Endogenous morphine-6-glucuronide (M6G) is present in the plasma of patients: validation of a specific anti-M6G antibody for clinical and basic research.

Endogenous morphine and its derivatives (morphine-6-glucuronide [M6G]; morphine-3-glucuronide [M3G]) are formed by mammalian cells from dopamine. Changes in the concentrations of endogenous morphine have been demonstrated in several pathologies (sepsis, Parkinson's disease, etc.), and they might be relevant as pathological markers. While endogenous morphine levels are detectable using enzyme-linked immunosorbant assay (ELISA), mass spectrometry (MS) analysis was, so far, the only approach to detect and quantify M6G. This study describes the preparation of a specific anti-M6G rabbit polyclonal antibody and its validation. The specificity of this antibody was assessed against 30 morphine-related compounds. Then, a M6G-specific ELISA-assay was tested to quantify M6G in the plasma of healthy donors, morphine-treated, and critically ill patients. The antibody raised against M6G displays a strong affinity for M6G, codeine-6-glucuronide, and morphine-3-6-glucuronide, whereas only weak cross-reactivities were observed for the other compounds. Both M6G-ELISA and LC-MS/MS approaches revealed the absence of M6G in the plasma of healthy donors (controls, n = 8). In all positive donors treated with morphine-patch (n = 5), M6G was detected using both M6G-ELISA and LC-MS/MS analysis. Finally, in a study on critically ill patients with circulating endogenous morphine (n = 26), LC-MS/MS analysis revealed that 73% of the positive-patients (19 of 26), corresponding to high M6G-levels in M6G-ELISA, contained M6G. In conclusion, we show that endogenous M6G can be found at higher levels than morphine in the blood of morphine-naive patients. With respect to the interest of measuring endogenous M6G in pathologies, we provide evidences that our ELISA procedure represents a powerful tool as it can easily and specifically detect endogenous M6G levels.

Vasostatin-I, a chromogranin A-derived peptide, in non-selected critically ill patients: distribution, kinetics, and prognostic significance.

PURPOSE: Chromogranin A (CGA) is released in the plasma during life-threatening illnesses. Its N-terminal 1-76 peptide, vasostatin-I (VS-I), has never been assessed in critically ill patients. Our aim was to examine whether the admission VS-I concentration has prognostic significance without having to specify a primary diagnosis. METHODS: VS-I concentrations were assessed with a new ELISA in 481 consecutive patients and 13 healthy controls. CGA and standard biological tests (including lactate) were performed; the simplified acute physiological score II (SAPS II) was calculated. Mortality was assessed at day 28. In a subgroup of 13 patients with shock, serial VS-I doses were given over 60 h. RESULTS: Critically ill patients had higher admission VS-I concentrations than controls [4.06 (2.78; 7.61) vs. 2.85 (2.47; 3.22) ng/ml, p < 0.001]. The plasma VS-I concentration was significantly lower in survivors than in non-survivors [3.70 (2.67; 6.12) vs. 5.75 (3.65; 11.20) ng/ml] and in the absence of shock [3.58 (2.59; 5.05) vs. 5.93 (3.30; 11.06) ng/ml, p < 0.001]. The survival rate was better in patients with VS-I concentrations under the median value of 3.97 ng/ml (p < 0.001). Admission VS-I and lactate values were independent predictors of mortality (p < 0.01). Moreover, taking them together, combined with age, provided a better indication for predicting mortality than taking each alone (p < 0.01). CONCLUSIONS: Significant amounts of VS-I are detected on admission in critically ill patients. A plasma VS-I concentration above 3.97 ng/ml is associated with poor outcome, and in routine practice simultaneous measurements of the three independent factors VS-I, lactate and age can affect the assessment of severity.