Separation of rotamers of 5-nitrosopyrimidines and estimation of binding constants of their complexes with ß-cyclodextrin by capillary electrophoresis.

For the first time, capillary electrophoresis has been successfully employed for the fast and highly efficient separations of a novel type of stereoisomers - planar rotamers (planamers) of four newly synthesized 5-nitrosopyrimidine derivatives. These derivatives can form two rotamers differing in the orientation of nitroso group due to strong intramolecular hydrogen bonds. Partial separation of rotamers of two 5-nitrosopyrimidines was achieved in alkaline 50 mM sodium tetraborate, pH 9.3, and in acidic 18.5/42 mM Tris/phosphate, pH 2.3, background electrolytes (BGEs) free of stereoselectors. To improve the separation of these rotamers and to attain the baseline or better separation of rotamers of other two 5-nitrosopyrimidines, various BGEs and different cyclodextrins-based stereoselectors were tested. The most effective, i.e. the fastest and baseline or better separations of rotamers of all analyzed 5-nitrosopyrimidines were achieved within a short time, 3.7-9.3 min, in the above alkaline or acidic BGEs using ß-cyclodextrin (ß-CD) or carboxymethyl-ß-CD as stereoselectors. Moreover, since the experiments with ß-CD resulted in good separations of rotamers of all the investigated 5-nitrosopyrimidines, the apparent binding constants of their complexes with this selector were determined from the dependence of their effective mobilities on the ß-CD concentration in the BGEs. The examined complexes were found to be relatively weak, with the apparent binding constants in the range 11.3-153.0 L/mol.

Procalcitonin Biomarker Kinetics to Predict Multiorgan Dysfunction Syndrome in Children With Sepsis and Systemic Inflammatory Response Syndrome.

BACKGROUND: Procalcitonin (PCT) kinetics is a good prognosis marker in infectious diseases, but few studies of children sepsis have been performed. OBJECTIVES: The aim of our study was to examine kinetics of procalcitonin, to evaluate its relationship with severity and to analyze its usefulness in the prediction of multiorgan dysfunction syndrome (MODS). PATIENTS AND METHODS: Prospective observational study in an 8-bed pediatric intensive care unit of a university hospital. Sixty-two children aged 0-19 years with systemic inflammatory response syndrome or septic states. The degree of severity was evaluated according pediatric logistic organ dysfunction (PELOD) score. Blood tests to determine levels of PCT were taken if the patients had the criteria of systemic inflammatory response syndrome or sepsis. The serum to determine levels of PCT in control group has been taken from patients undergoing elective surgery. RESULTS: Higher values of PCT were identified in patients with PELOD score 12 and more compared to those with PELOD < 12 (P = 0.016). Similarly, higher PCT values were found in patients who developed MODS in contrast to those without MODS (P = 0.011). According to ROC analysis cut-off value of 4.05 ng/mL was found to best discriminate patients with PELOD < 12 and PELOD ≥ 12 with AUC = 0.675 (P = 0.035). Effect of procalcitonin levels on mortality was not demonstrated. CONCLUSIONS: Levels of procalcitonin from day 1 to day 5 are related to the severity and multiorgan dysfunction syndrome in children.

Trefoil factor 3 as a marker of intestinal cell damage during sepsis.

OBJECTIVE: Gastrointestinal dysfunction or gut failure frequently occurs in seriously ill patients and can be responsible for multi-organ failure. Trefoil factor 3 (TFF3) was characterized for its role in reconstitution of an epithelial barrier after mucosal injury in the jejunum. The aims of our study was an analysis of TFF3 levels dynamics in patients with sepsis and the correlation of TFF3 with severity of sepsis and mortality. METHODS: Prospective observational study, a ten days evaluation period in children aged 0-19 years with systemic inflammatory response syndrome or septic state. Blood tests to determine levels of TFF3 were obtained as long as the patient met the criteria for systemic inflammatory response syndrome or sepsis. RESULTS: Analysis of dynamics revealed steady levels of TFF3 during the 10 day period evaluated. TFF3 levels could not differentiate between various septic conditions in patients until a marked organ dysfunction developed. Higher Area Under Curve was noticed between control group and patients with sepsis. We could not make any strong conclusions based on mortality model. CONCLUSIONS: Levels of TFF3 are elevated in paediatric patients with sepsis through organ dysfunction.

Trefoil factor 2 expression and its significance as a predictor of severity of sepsis in children.

Intestinal injury significantly contributes to critical illness, sepsis and multiorgan failure. TFF2 (Trefoil Factor 2) is expressed and secreted preferentially by gastric mucous neck cells. TFF2 gene expression is promptly increased after gut injury, and its expression profile broadens to include the regenerative epithelia of virtually the entire gastrointestinal tract. The first objective of our study was an analysis of TFF2 levels dynamics in patients with Systemic Inflammatory Response Syndrome (SIRS) or septic condition during a 5-day period after admission. The second objective was to determine optimal cut-off value and quantify diagnostic characteristics of TFF2 between controls and patients with various septic states. The study included 57 children aged 0-19 years, with expected or proven SIRS and septic condition. The degree of severity was evaluated according to PELOD Score (Pediatric Logistic Organ Dysfunction). Blood samples to determine levels of TFF2 factor were taken during the time patient met the criteria of SIRS or sepsis. Control group samples to determine the serum levels of TFF2 were taken from patients undergoing elective surgery. Analysis of TFF2 levels dynamics revealed that TFF2 levels kept steady state during the 5-day period. Significantly higher levels of TFF2 were in patients with Multiple Organ Dysfunction Syndrome (MODS). The difference was noticed also in ROC analysis.

Trefoil factor 1 as a marker of mucosal damage of the gastrointestinal tract in children with sepsis.

Intestinal ischemia and reperfusion is a common pathway for many diseases in children. The objective of our study was an analysis of Trefoil factor 1 levels dynamics in patients with SIRS or septic condition during a 5-day period. Analysis of TFF1 levels dynamics revealed that TFF1 levels kept steady state during the 5-day period. TFF1 levels were similar in patients with SIRS, sepsis and severe sepsis. Significantly higher levels of TFF1 were in patients with septic shock and MODS.

Classification of infections in intensive care units: a comparison of current definition of hospital-acquired infections and carrier state criterion.

BACKGROUND: The rate of nosocomial infection appears to depend on whether it is calculated using the Center for Disease Control (CDC) or carrier state criteria. The objective of this study was to differentiate between primary endogenous (PE), secondary endogenous (SE) and exogenous (EX) infections, and to compare this classification with CDC criteria for nosocomial infections. METHODS: Children hospitalized for more than 72 h at pediatric intensive care unit during 2004-2005 were enrolled. Children, who had the infection before the admission, and or did not develop an infection within the hospitalization were excluded. Surveillance samples were sampled on admission, and then twice a week. Diagnostic samples were obtained when infection was suspected based on the clinical condition and laboratory findings. Infections were evaluated as PE, SE and EX, and their incidences were compared with CDC criteria for nosocomial infections. RESULTS: One hundred seventy eight patients were enrolled in the study. Forty-four patients (24.7%) develop infection. Twenty-seven patients (61.3%) had PE, 10 patients (22.7%) had SE, and 7 patients (15.9%) had EX infection. Secondary endogenous and EX infections are considered as nosocomial, thus 17 patients (38.6%) had a nosocomial infection. Thirty-one patients (70.5%) met CDC criteria for nosocomial infections. Seventeen patients (55%) were classified as PE, and 14 patients (45%) as SE or EX infections. CONCLUSION: Seventy percent of infections (31 out of 44 patients) met the CDC criteria for nosocomial infections, but only 39% of infections (17 out of 44 patients) were classified as nosocomial based on carrier state classification.

The usefulness of S100B, NSE, GFAP, NF-H, secretagogin and Hsp70 as a predictive biomarker of outcome in children with traumatic brain injury.

BACKGROUND: Predicting the long-term outcome after traumatic brain injury (TBI) is an important component of treatment strategy. Despite dramatically improved emergency management of TBI and apparent clinical recovery, most patients with TBI still may have long-term central nervous system (CNS) impairment. METHODS: Sixty-three patients with TBI were enrolled into the prospective study. Venous blood samples were taken at admission and every 24 h for a maximum of 6 consecutive days. Serum concentrations of the biomarkers S100B, neuron-specific enolase (NSE), GFAP, NF-H, secretagogin and Hsp70 were quantified immuno-luminometrically or by enzyme-linked immunosorbent assay. The outcome was evaluated 6 months after TBI using the Glasgow Outcome Scale (GOS) in all patients. RESULTS: The S100B levels in patients with worse outcome (GOS 4 or death) were already significantly higher at D0 (p < 0.001; p = 0.002). NSE levels were significantly higher in patients who died or had worse outcomes (p < 0.001; p = 0.003). Patients who had worse outcomes (GOS) or died had higher GFAP values (p < 0.001; p < 0.001), but their dynamics were similar over the same period. NF-H grew significantly faster in patients who had a worse GOS or died (p < 0.001; p = 0.001). CONCLUSIONS: Although further prospective study is warranted, these findings suggest that levels of biomarkers correlate with mortality and may be useful as predictors of outcome in children with TBI.

Dynamics of glial fibrillary acidic protein during traumatic brain injury in children.

BACKGROUNDS: Glial fibrillary acidic protein (GFAP) is a monomeric intermediate filament protein found in the astroglial cytoskeleton and is not found outside the central nervous system. It is a brain-specific protein that is released after traumatic brain injury (TBI). METHODS: This prospective study enrolled 59 children who had TBI, as verified by computed tomography. Daily GFAP measurement began at admission (<12 hours after trauma) and continued for 6 days. Blood samples were analyzed for GFAP by enzyme-linked immunosorbent assay. Outcome was assessed using the Glasgow Outcome Scale (GOS) at 6 months after injury. RESULTS: The median serum levels of GFAP at admission were 7.47 ng/mL in patients who died, compared with 0.12 ng/mL in patients who survived (p=0.002). GFAP levels were significantly higher in patients who had a poor outcome 6 months after injury than in those who were alive or had good outcome (p<0.001). The area under the receiver operating characteristic curve for GFAP was 0.833 for day 0 and 0.884 for day 2. CONCLUSIONS: These results suggest that determination of serum levels of GFAP may add to the clinical assessment of the primary damage and prediction of outcome after severe TBI.

Hyperphosphorylated neurofilament NF-H as a predictor of mortality after brain injury in children.

OBJECTIVE: The aim of the study was to determine whether serum levels of hyperphosphorylated neurofilament NF-H correlate with severity of brain injury in children. METHODS: Forty-nine patients with traumatic brain injury (TBI) were enrolled into the prospective study. Venous blood samples were taken after admission and every 24 h for a maximum of 6 consecutive days. Serum NF-H concentrations were quantified by enzyme-linked immunosorbent assay. The outcome was evaluated 6 months after TBI using Glasgow Outcome Scale (GOS) in all patients. RESULTS: The quantitative level of pNF-H remained significantly higher in patients with poor outcome (GOS = 1) in comparison with the other patients for the 2nd-4th day (p = 0.027; p = 0.019; p = 0.01). Levels of pNF-H were significantly higher in patients with diffuse axonal injury on initial CT scan (p = 0.004). Normal levels pNF-H in the paediatric population are unknown. Objective ROC analysis was identification of optimal cut-offs of proteins for prediction of GOS = 1. CONCLUSIONS: Although further, prospective study is warranted, these findings suggest that levels of hyperphosphorylated neurofilament NF-H correlate with mortality and may be useful as predictors of outcome in children with TBI.