Simona Storti

Genetic polymorphisms in folate and homocysteine metabolism as risk factors for DNA damage

Epidemiological studies indicated a role for polymorphisms in genes of folate and homocysteine (Hcy) metabolism in the etiology of neurodegenerative disease, congenital defects and coronary artery disease (CAD). This study investigated the effect of several polymorphisms [C677 T, A1298C of methylenetetrahydrofolate reductase (MTHFR) and A66G of methionine synthase reductase (MTRR) genes] on Hcy levels and DNA damage in 68 patients who underwent coronary angiography. Plasma Hcy concentrations were higher in patients with multivessel disease with respect to monovessel disease and no-CAD patients (19.4±2.6 vs 11.6±1.2 and 13.7±1.4 μmol/l, respectively; P=0.03). 677TT patients had higher Hcy levels than those with 677CC or 677CT genotypes (26.2±4.3 vs 13.1±1.4 and 13.0±1.4 μmol/l, respectively; P=0.0006). No significant associations were found between A1298C and A66G polymorphisms and plasma Hcy levels. Among patients with 677CC genotype, 66GG individuals tended to have higher levels of Hcy than 66AA homozygotes (14.5±1.9 vs 8.9±0.7 μmol/l, P=0.06). Multivessel disease patients showed an increased frequency of DNA damage, measured by the micronucleus (MN) frequency, as compared to monovessel disease and no-CAD subjects (12.5±1.1 vs 8.5±0.8 and 8.2±0.9, respectively; P=0.006). The MN were positively correlated with Hcy levels (r=0.33, P=0.006) and were significantly higher in subjects with the 677TT genotype compared with the 677CC or 677CT genotypes (14.4±2.0 vs 8.8±1.2 and 9.5±0.7, respectively; P=0.006). A1298C and A66G polymorphisms had no effect on MN frequency. However, among 677TT patients, 66GG subjects tended to have higher levels of MN than those 66AG and 66AA (18.2±3.6 vs 13.8±4.0 and 10.3±1.7, respectively; P=NS). Our results indicate that genetic instability may be associated with increased risk for multiple Hcy-related diseases.

Association between 5,10-Methylenetetrahydrofolate Reductase C677T and A1298C Polymorphisms and Conotruncal Heart Defects

Abstract Reports related some polymorphisms of the 5,10-methylenetetrahydrofolate reductase (MTHFR) to folate-dependent neural tube defects. In view of the common origin of the cells involved both in neural tube closure and heart septation, we analyzed the MTHFR C677T and A1298C polymorphisms in mothers of children with conotruncal heart defect (CD) and in their offspring to evaluate the association between the MTHFR genotype and the risk of CD. We genotyped 103 Italian mothers with CD offspring, 200 control mothers, 103 affected children and their fathers by restriction fragment length polymorphism analysis. No increased risk was observed for the prevalence of the 677TT genotype by itself in affected children and in their mothers. The combined maternal 677TT/1298AA and 677CC/1298CC genotypes have odds ratio of 1.73 and 1.85, respectively. The prevalence of 1298CC genotype in the affected children gives odds ratio of 1.90, that becomes 2.31 for the 677CC/1298CC genotype. However, none of the odds ratios was statistically significant. We observed a higher frequency of the 677T allele in Italy than in other European countries. No association has been demonstrated between the 677TT MTHFR genotype and CD.

Comparison between analytical performances of polyclonal and monoclonal electrochemiluminescence immunoassays for NT-proBNP

BACKGROUND We evaluated the analytical characteristics of electrochemiluminescence (ECLIA) immunoassay for NT-proBNP (proBNP II, Roche Diagnostics, Germany) and compared its analytical performance to that of the previous polyclonal method. METHODS We measured NT-proBNP in EDTA plasma samples of 177 consecutive cardiac patients (69 females and 108 males; mean age 62.2+/-16.4 years, range 13 to 96 years) with monoclonal and polyclonal ECLIA methods following manufacturers instructions using an Elecsys 2010 analyzer. RESULTS Monoclonal ECLIA method for NT-proBNP assay showed an imprecision (CV%) lower than 3% at the cut-off value (i.e., 150 ng/L). No significant interference was found in plasma samples containing high levels of hemoglobin, triglycerides or bilirubin. EDTA plasma showed slightly, but significantly lower NT-proBNP values than serum (on average -6.3%) and lithium-heparinized plasma (on average -3.9%) samples. Finally, a very close linear regression was found between the NT-proBNP values found by either monoclonal or polyclonal ECLIA method (monoclonal ECLIA=-72.17+1.04 polyclonal ECLIA, n=177, R=0.993). CONCLUSIONS The monoclonal ECLIA method showed very similar analytical characteristics with slightly lower NT-proBNP results (on average -2.5%) than the polyclonal ECLIA method. The difference between monoclonal and polyclonal methods seems to be too slight to change the reference range and decisional values for NT-proBNP assay, as previously assessed by the polyclonal ECLIA method.

Free triiodothyronine: a novel predictor of postoperative atrial fibrillation

OBJECTIVE Despite improved perioperative management, atrial fibrillation (AF) after coronary artery bypass grafting (CABG) remains a relevant clinical problem, whose pathogenetic mechanisms remain incompletely explained. A reduced incidence of postoperative AF has been described in CABG patients receiving IV tri-iodothyronine (T3). This study was designed to define the role of thyroid metabolism on the genesis of postoperative AF. METHODS AND RESULTS Free T3 (fT3), free thyroxine (fT4), and thyroid stimulating hormone were assayed at admission in 107 consecutive patients undergoing isolated CABG surgery. Patients with thyroid disease or taking drugs known to interfere with thyroid function were excluded. A preoperative rhythm other than sinus rhythm was considered an exclusion criterion. Thirty-three patients (30.8%) had postoperative AF. An older age (P=0.03), no therapy with beta-blockers (P=0.08), chronic obstructive pulmonary disease (P=0.08), lower left ventricle ejection fraction (P=0.09) and lower fT3 concentration (P=0.001), were univariate predictors of postoperative AF. On multivariate analysis, low fT3 concentration and lack of beta-blocking therapy were independently related with the development of postoperative AF (odds ratio, OR, 4.425; 95% confidence interval, CI, 1.745-11.235; P=0.001 and OR 3.107; 95% CI 1.087-8.875; P=0.03, respectively). Postoperative AF significantly prolonged postoperative hospital stay (P=0.002). CONCLUSIONS Low basal fT3 concentration can reliably predict the occurrence of postoperative AF in CABG patients.

Reference intervals for brain natriuretic peptide in healthy newborns and infants measured with an automated immunoassay platform

Abstract Background: In order to assess the reference intervals for B-type natriuretic hormone (BNP) in the first days of life, we measured peptide concentrations using the fully automated Access platform. Methods: Plasma BNP was measured in 188 apparently healthy newborns and infants throughout the first month of extra-uterine life, as well as in 245 healthy infants ranging from 1 month to 12 years of age. Results: BNP showed the highest concentrations in the first 2 days of life, with a progressive decline afterwards. Moreover, BNP values in the first week of life were significantly higher (p<0.0001) than values observed in the next periods. As a result, a significant negative correlation was found between BNP and age values when considering all 433 samples (ρ=–0.816, p<0.0001 by the Spearman rank correlation test). There was no significant difference between BNP values found in males and females. Conclusions: According to this data, our study indicates that at least two reference intervals should be used for newborns and infants. The first, with higher BNP values for neonates in the first week of extra-uterine life, and the other, with lower BNP values for infants aged 2 weeks to 12 years. Clin Chem Lab Med 2010;48:697–700.

Analytical performance and clinical results of a fully automated MEIA system for brain natriuretic peptide assay: comparison with a point of care testing method

Abstract The aim of this study was to evaluate the analytical performance of a recently available immunoassay for brain natriuretic peptide (BNP), based on microparticle enzyme immunoassay (MEIA, AxSYM System, Abbott Laboratories), whose analytical characteristics and clinical results were compared with those of a point of care testing (POCT) method (TRIAGE system, Biosite Diagnostics). The within-run and total imprecision of the MEIA system were 18.4% and 19.8% at 21 ng/l, 8.0% and 14.8% at 183 ng/l, and 5.7% and 14.0% at 319 ng/l, respectively. The detection limit of the MEIA system was tested by repeatedly measuring (n = 20) the 0 calibrator in four different runs; a mean +3 SD value of 5.6 ± 4.8 ng/l (range 1.8–12.6 ng/l) was obtained. A close linear relationship (MEIA = –22.5 + 1.71 POCT method, R = 0.950, n = 296) was found (BNP concentration: 5–5500 ng/l), with a significant bias (mean difference: 164.8 ng/l, p < 0.0001). Mean BNP concentration measured in 94 reference subjects (57 women and 37 men; mean age 43.5 ± 14.0 years) was higher with MEIA than POCT, (25.9 ± 32.7 ng/l vs. 11.7 ± 8.9 ng/l, p < 0.0001). The same trend was observed also in 202 cardiac patients (620.6 ± 1082.2 ng/l vs. 386.1 ± 594.5 ng/l, p < 0.0001). Our data suggest that MEIA and POCT have quite similar analytical performance but different clinical results. Then, different reference values, as well as cut-off values, should be taken into account for the clinical use of these two immunoassays.

The combined use of neutrophil gelatinase-associated lipocalin and brain natriuretic peptide improves risk stratification in pediatric cardiac surgery.

Abstract Background: The aim of this study is to test the hypothesis whether the combined use of a cardio-specific biomarker, the brain natriuretic peptide (BNP) and a marker of early renal damage, the assay of urinary neutrophil gelatinase-associated lipocalin (uNGAL), may improve risk stratification in pediatric cardiac surgery. Methods: We prospectively enrolled 135 children [median age 7 (interquartile range 1–49) months] undergoing to cardiac surgery for congenital heart disease. All biomarkers were evaluated pre- and post-operatively at different times after cardiopulmonary-bypass (CPB): uNGAL at 2, 6 and 12 h; BNP at 12 and 36 h; serum creatinine at 2, 6, 12, and 36 h. Primary endpoints were development of acute kidney injury (AKI) (defined as 1.5 serum creatinine increase) and intubation time. Results: AKI occurred in 39% of patients (65% neonates and 32% older children, p=0.004). The peak of uNGAL values occurred more frequently at 2 h. uNGAL values at 2 h [median 28.2 (interquartile range 7.0–124.6) ng/L] had a good diagnostic accuracy for early diagnosis of AKI with an AUC (area under the curve) ROC (receiver operating characteristic) curve of 0.85 (SE 0.034). Using multivariable logistic regression analysis, development of AKI was significantly associated with uNGAL values at 2 h after CPB [OR=1.88 (1.30–2.72, p=0.001)], together with the CPB time and Aristotle score, as an index of complexity of the surgical procedure, while pre-operative BNP values were not. Furthermore, uNGAL and pre-operative BNP values (together with Aristotle score) were significantly associated with adverse outcome (longer intubation time and mortality). Conclusions: Pre-operative BNP and uNGAL values after surgery (together with the Aristotle score) were independently associated with a more severe course and worse outcome in children undergoing cardiac surgery for congenital heart disease.

Diagnostic accuracy of B-type natriuretic hormone for congenital heart disease in the first month of life

Abstract Background: The goal of the present study was to evaluate the diagnostic accuracy of B-type natriuretic hormone (BNP) assay in children with congenital heart disease (CHD) in the first month of life. Methods: BNP was measured in 152 neonates with CHD; 154 healthy children matched for age were used as controls. BNP was measured with a fully automated platform (Triage BNP reagents, Access Immunoassay Systems, Beckman Coulter, Inc., Fullerton, CA, USA). Results: BNP values were significantly higher (p<0.0001) in newborns and infants with CHD compared with control (CHD patients: median 1167.5 ng/L, range 25–54,447 ng/L; healthy children: median 150.5 ng/L, range 5–866 ng/L). The diagnostic accuracy of BNP was assessed using the receiver operating characteristic (ROC) analysis, taking into account the three different groups divided according to age. Group 1: all CHD patients and healthy newborns and infants as a whole (i.e., from birth to the 30th day of life); Group 2: from the 1st to 3rd day of life; Group 3: from the 4th to 30th day of life. The area under the curve (AUC) of the ROC curve for Group 3 (0.935) was significantly higher than that for Group 1 (0.843, p=0.009) and Group 2 (0.769, p=0.0003), while the AUC values of Group 1 and Group 2 were not significantly different (p=0.191). Conclusions: BNP may be considered a useful marker for screening in the integrated approach of newborns, infants and children with suspected CHD. However, the accuracy of the BNP assay varies greatly during the first month of extra-uterine life, showing the lowest diagnostic accuracy in the first 3 days after birth. After the second week of life, the biomarker becomes more accurate in ruling in CHD. Clin Chem Lab Med 2010;48:1333–8.

Reference values for plasma B-type natriuretic peptide in the first days of life.

The clinical relevance of B-type natriuretic peptide (BNP)1 and the amino terminal fragment of its prohormone (NT-proBNP) as biomarkers in pediatric heart disease have recently been shown (1)(2). BNP and NT-proBNP concentrations are dependent on age and sex, at least in adults(3). Because commercial methods are affected differently by the presence in plasma of several peptides derived from the degradation of intact prohormone and BNP(3), little if any agreement exists among reported reference intervals, especially those used for infants during the first days of extrauterine life(1). Data are scarce regarding the reference values for BNP and NT-proBNP in infancy (1)(2)(4). Recently, Nir et al.(2) summarized NT-proBNP concentrations measured with an electrochemiluminescence immunoassay method in 690 healthy individuals (47% males) with ages ranging from birth to 18 years, including 127 newborns in the first week of life (43 in the first 2 days). The NT-proBNP concentrations were highest in the first days of life, and then showed a marked decline. Concentrations in males and females differed only for children age 10–14 years. Large reference value studies for BNP for newborns and infants have …

Reference values for urinary neutrophil gelatinase-associated lipocalin (NGAL) in pediatric age measured with a fully automated chemiluminescent platform

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as the most promising biomarker of acute kidney injury. However, there are no reliable data on analytical sensitivity and reference limits of urinary NGAL (uNGAL) assay in pediatric age. The aim of the present study is to evaluate the analytical sensitivity and the reference range of uNGAL measured in urine specimens of pediatric age with the fully automated platform ARCHITECT® i1000. Methods: A total of 333 urine samples were collected from 25 healthy newborns (16 males and 9 females; age 1–4 days) and 308 children (150 males and 147 females; mean age 80.7 months, range 0.63–248 months) and assayed for uNGAL by two different Italian centers (Department of Laboratory Medicine of the Fondazione Toscana G. Monasterio of Pisa and Massa and the Clinical Pathology Laboratory Unit of Istituto Giannina Gaslini of Genova). Results: The calculated limits of blank (LOB) and detection (LOD) values were 0.5 ng/mL and 0.95 ng/mL, respectively. The distribution of uNGAL values approximated a log-normal distribution (median 5.2 ng/mL, interquartile range 2.5–12.8 ng/mL, 99th percentile 117.6 ng/mL). uNGAL values of the 25 neonates were significantly higher than those of 308 children (neonates: mean 44.2 ng/mL, median 30.3 ng/mL, range 5.2–137.4 ng/mL; children: mean 10.2 ng/mL, median 4.6 ng/mL, range 0.2–146.7 ng/mL; p<0.0001). Conclusions: uNGAL assay shows a good analytical sensitivity and imprecision, which allows the measurement of uNGAL values around the cut-off value (i.e., 117.6 ng/mL) with an imprecision <5 CV%. The distribution of uNGAL values in pediatric age approximates a log-normal distribution, with values which are higher in neonates compared to children.