Remigio Moratti

Serum N-Terminal Pro–Brain Natriuretic Peptide Is a Sensitive Marker of Myocardial Dysfunction in AL Amyloidosis

Background—Brain natriuretic peptide (BNP) is a marker of ventricular dysfunction and can be used to assess prognosis in heart failure and after myocardial infarction. Heart involvement is the most important prognostic factor and causes death in almost all patients with light-chain amyloidosis (AL). We investigated the prognostic value of NT-proBNP and its utility in monitoring amyloid heart dysfunction. Methods and Results—NT-proBNP was quantified at diagnosis in 152 consecutive patients seen at the coordinating center of the Italian Amyloidosis Study Group (Pavia) from 1999 throughout 2001. Heart involvement was estimated on the basis of clinical signs, electrocardiography, and echocardiography. NT-proBNP concentrations differed in patients with (n=90, 59%) and without (n=62, 41%) heart involvement (median: 507.8 pmol/L versus 22.1 pmol/L, P =10−7). The best cutoff for heart involvement was at 152 pmol/L (sensitivity: 93.33%, specificity: 90.16%, accuracy: 92.05%) and distinguished two groups with different survival (P <0.001). The Cox multivariate model including NT-proBNP was better than models including echocardiographic and clinical signs of heart involvement. NT-proBNP appeared to be more sensitive than conventional echocardiographic parameters in detecting clinical improvement or worsening of amyloid cardiomyopathy during follow-up. Conclusions—NT-proBNP appeared to be the most sensitive index of myocardial dysfunction and the most powerful prognostic determinant in AL amyloidosis. It adds prognostic information for newly diagnosed patients and can be useful in designing therapeutic strategies and monitoring response. NT-proBNP is a sensitive marker of heart toxicity caused by amyloidogenic light chains.

The combination of high-sensitivity cardiac troponin T (hs-cTnT) at presentation and changes in N-terminal natriuretic peptide type B (NT-proBNP) after chemotherapy best predicts survival in AL amyloidosis

In light-chain (AL) amyloidosis, prognosis is dictated by cardiac dysfunction. N-terminal natriuretic peptide type B (NT-proBNP) and cardiac troponins (cTn) are used to assess the severity of cardiac damage. We evaluated the prognostic relevance of a high-sensitivity (hs) cTnT assay, NT-proBNP, and cardiac troponin I in 171 consecutive patients with AL amyloidosis at presentation and 6 months after treatment. Response and progression of NT-proBNP were defined as more than 30% and more than 300 ng/L changes. All 3 markers predicted survival, but the best multivariable model included hs-cTnT. The hs-cTnT prognostic cutoff was 77 ng/L (median survival 10.6 months for patients with hs-cTnT above the cutoff). After treatment, response and progression of NT-proBNP and a more than 75% increase of hs-cTnT were independent prognostic determinant. In AL amyloidosis, hs-cTnT is the best baseline prognostic marker. Therapy should be aimed at preventing progression of cardiac biomarkers, whereas NT-proBNP response confers an additional survival benefit.

Identification of Amyloidogenic Light Chains Requires the Combination of Serum-Free Light Chain Assay with Immunofixation of Serum and Urine

BACKGROUND The diagnosis of systemic immunoglobulin light-chain (AL) amyloidosis requires demonstration of amyloid deposits in a tissue biopsy and amyloidogenic monoclonal light chains. The optimal strategy to identify the amyloidogenic clone has not been established. We prospectively assessed the diagnostic sensitivity of the serum free light chain (FLC) kappa/lambda ratio, a commercial serum and urine agarose gel electrophoresis immunofixation (IFE), and the high-resolution agarose gel electrophoresis immunofixation (HR-IFE) developed at our referral center in patients with AL amyloidosis, in whom the amyloidogenic light chain was unequivocally identified in the amyloid deposits. METHODS The amyloidogenic light chain was identified in 121 consecutive patients with AL amyloidosis by immunoelectron microscopy analysis of abdominal fat aspirates and/or organ biopsies. We characterized the monoclonal light chain by using IFE and HR-IFE in serum and urine and the FLC kappa/lambda ratio in serum. We then compared the diagnostic sensitivities of the 3 assays. RESULTS The HR-IFE of serum and urine identified the amyloidogenic light chain in all 115 patients with a monoclonal gammopathy. Six patients with a biclonal gammopathy were omitted from the statistical analysis. The diagnostic sensitivity of commercial serum and urine IFE was greater than that of the FLC kappa/lambda ratio (96% vs 76%). The combination of serum IFE and the FLC assay detected the amyloidogenic light chain in 96% of patients. The combination of IFE of both serum and urine with the FLC kappa/lambda ratio had a 100% sensitivity. CONCLUSIONS The identification of amyloidogenic light chains cannot rely on a single test and requires the combination of a commercially available FLC assay with immunofixation of both serum and urine.

A staging system for renal outcome and early markers of renal response to chemotherapy in AL amyloidosis

The kidney is involved in 70% of patients with immunoglobulin light-chain (AL) amyloidosis, but little is known on progression or reversibility of renal involvement, and criteria for renal response have never been validated. Newly diagnosed patients from the Pavia (n = 461, testing cohort) and Heidelberg (n = 271, validation cohort) centers were included. Proteinuria >5 g/24 h and estimated glomerular filtration rate (eGFR) <50 mL/min predicted progression to dialysis best. Proteinuria below and eGFR above the thresholds indicated low risk (0 and 4% at 3 years in the testing and validation cohorts, respectively). High proteinuria and low eGFR indicated high risk (60% and 85% at 3 years). At 6 months, a ≥25% eGFR decrease predicted poor renal survival in both cohorts and was adopted as criterion for renal progression. A decrease in proteinuria by ≥30% or below 0.5 g/24 h without renal progression was the criterion for renal response, being associated with longer renal survival in the testing and validation populations. Hematologic very good partial or complete remission at 6 months improved renal outcome in both populations. We identified and validated a staging system for renal involvement and criteria for early assessment of renal response and progression in AL amyloidosis that should be used in clinical practice and trial design.

Oxidation of Low-Density Lipoprotein in Atherosclerosis from Basic Biochemistry to Clinical Studies

Although it has been known for long time that atherosclerosis is associated with lipid deposition, only recently it has been accepted that the plasmatic concentration of cholesterol, especially LDL cholesterol, is a risk factor for atherosclerosis. However, chemically modified LDL, but not native LDL, is able to induce the formation of foam cells, the hallmark of atherosclerosis. LDL oxidation is likely to be the most important form of LDL modification in humans. In biochemical terms, LDL oxidation is a free radical driven chain reaction where polyunsaturated fatty acids are converted to lipid peroxides, which easily decompose to many products, including biologically active aldehydes. The assay of LDL oxidation in biological fluids is problematic; direct assays detect a product of LDL oxidation whereas indirect assays give an indicator of LDL oxidation susceptibility. In general, epidemiological studies support the concept that the level of plasmatic lipophilic antioxidants, tocopherols and carotenoids, is low in populations at increased risk for atherosclerosis. However, clinical trials based on vitamin E as antioxidant showed inconclusive results, suggesting that supplementation with vitamin E is not generically recommended for atherosclerotic patients. These results, however, do not contradict that oxidation of lipoprotein is involved in atherosclerosis; rather, this negative outcome raises a number of considerations such as the need for a reliable marker of lipoprotein oxidation in plasma and a more complete information about the physiological triggers of lipoprotein oxidation.

Biological Variation of N-Terminal Pro-Brain Natriuretic Peptide in Healthy Individuals

Brain natriuretic peptide (BNP) and its N-terminal prohormone (NT-proBNP) fragment have been shown to be effective in diagnosing left ventricular dysfunction (1)(2), and in particular, they have a strong negative predictive value (3). NT-proBNP and the hormone are secreted on an equimolar basis, but NT-proBNP lacks a clearance receptor. It therefore has a longer half-life in serum than the active hormone does, and its circulating concentration is believed to be less influenced by the conditions under which the blood sample is taken. Information on the biological variation of NT-proBNP is not available; this is limiting because the clinical utility of laboratory data can be affected by physiologic variation (4). Here we report the results of …

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood.

Best use of cardiac biomarkers in patients with AL amyloidosis and renal failure.

In AL amyloidosis prognosis depends on the severity of heart dysfunction which is best assessed by natriuretic peptides (BNP and NT‐proBNP). However, their clearance relies on glomerular filtration rate (GFR) and their concentration increases with renal failure. We evaluated the diagnostic and prognostic performance of NT‐proBNP and BNP in 248 patients with AL amyloidosis with different degrees of renal failure. Patients were grouped according to GFR. Group 1 comprised 109 patients with GFR ≥60 mL/min/1.73 m2, Group 2, 77 subjects with GFR <60 and ≥15 mL/min/1.73 m2, and Group 3, 62 patients with GFR <15 mL/min/1.73 m2. The ability of natriuretic peptides to detect heart involvement and to predict survival in the three groups was assessed. Decreasing eGFR required higher cutoffs of both NT‐proBNP and BNP for detecting heart involvement and predicting survival. Both natriuretic peptides were independent prognostic markers in Groups 1 and 2, whereas in Group 3 only BNP independently predicted survival. Natriuretic peptides are powerful and useful markers of cardiac dysfunction and prognosis, provided that eGFR is considered in interpreting their clinical meaning. BNP should be preferred in patients with end‐stage renal failure. Am. J. Hematol. 87:465–471, 2012.

Comparison of brain natriuretic peptide (BNP) and NT-proBNP in screening for pulmonary arterial hypertension in patients with systemic sclerosis

Objective. To compare the performance of brain natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) in screening for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc). Methods. Between January 2008 and March 2009, outpatients referred to our unit and satisfying LeRoy criteria for SSc were assessed for PAH. Doppler echocardiography, BNP measurement, and NT-proBNP measurement were done concomitantly for a complete clinical, instrumental, and biochemical evaluation. Right-heart catheterization was carried out in cases of suspected PAH [estimated pulmonary arterial pressure (PAP) ≥ 36 mm Hg; diffusion capacity for carbon monoxide (DLCO) ≤ 50% of predicted value; 1-year DLCO decline ≥ 20% in absence of pulmonary fibrosis; unexplained dyspnea]. Results. One hundred thirty-five patients were enrolled (124 women, 11 men; 96 limited SSc, 39 diffuse SSc); precapillary PAH was found in 20 patients (15 limited SSc, 5 diffuse SSc). The estimated PAP correlated with both BNP (R = 0.3; 95% CI 0.14–0.44) and NT-proBNP (R = 0.3, 95% CI 0.14–0.45). BNP [area under the curve (AUC) 0.74, 95% CI 0.59–0.89] was slightly superior to NT-proBNP (AUC 0.63, 95% CI 0.46–0.80) in identification of PAH, with diagnosis cutoff values of 64 pg/ml (sensitivity 60%, specificity 87%) and 239.4 pg/ml (sensitivity 45%, specificity 90%), respectively. BNP (log-transformed, p = 0.032) and creatinine (p = 0.049) were independent predictors of PAH, while NT-proBNP was not (p = 0.50). Conclusion. In our single-center study, the performance of BNP was slightly superior to that of NT-proBNP in PAH screening of patients with SSc, although normal levels of these markers do not exclude diagnosis. We observed that impaired renal function is associated with an increased risk of PAH in SSc. Further multicenter studies are needed to confirm our results (ClinicalTrials.gov ID NCT00617487).

Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 microm, 150 x 4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate-acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9+/-4.0% for non-deproteinized samples and 93.2+/-4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean+/-SD concentrations in the darkness period were 23.18+/-7.44 pg/ml for free melatonin and 82.5+/-36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23+/-2.22 and 7.40+/-5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01+/-2.31 and 28.55+/-6.95 pg/ml for the free and total fraction, respectively.