Andrey N. Kara

Human Pro–B-Type Natriuretic Peptide Is Processed in the Circulation in a Rat Model

BACKGROUND The appearance of B-type natriuretic peptide (BNP) in the blood is ultimately caused by proteolytic processing of its precursor, proBNP. The mechanisms leading to the high plasma concentration of unprocessed proBNP are still poorly understood. The goals of the present study were to examine whether processing of proBNP takes place in the circulation and to evaluate the clearance rate of proBNP and proBNP-derived peptides. METHODS We studied the processing of human proBNP in the circulation and the clearance rate of proBNP and proBNP-derived peptides (BNP and N-terminal fragment of proBNP, NT-proBNP) in rats by injecting the corresponding peptides and analyzing immunoreactivity at specific time points. Glycosylated and nonglycosylated proBNP and NT-proBNP were used in the experiments. We applied immunoassays, gel filtration, and mass spectrometry (MS) techniques to analyze the circulation-mediated processing of proBNP. RESULTS ProBNP was effectively processed in the circulation into BNP (1-32) and various truncated BNP forms as confirmed by gel filtration and MS analysis. Glycosylation of proBNP close to the cleavage-site region suppressed its processing in the circulation. The terminal half-life for human glycosylated proBNP was 9.0 (0.5) min compared with 6.4 (0.5) min for BNP. For NT-proBNP, the terminal half-lives were 15.7 (1.4) min and 15.5 (1.3) min for glycosylated and nonglycosylated forms, respectively. CONCLUSIONS In rats, processing of human proBNP to active BNP occurs in the circulation. The clearance rate of proBNP is quite similar to that of BNP. These observations suggest that peripheral proBNP processing may be an important regulatory step rather than mere degradation.

Anti–Cardiac Troponin Autoantibodies Are Specific to the Conformational Epitopes Formed by Cardiac Troponin I and Troponin T in the Ternary Troponin Complex

BACKGROUND Autoantibodies to cardiac troponins (TnAAbs) could negatively affect cardiac troponin I (cTnI) measurements by TnAAbs-sensitive immunoassays. We investigated the epitope specificity of TnAAbs and its influence on cTnI immunodetection in patients with acute myocardial infarction (AMI). METHODS The specificity of TnAAbs was studied in immunoassays and gel-filtration experiments. The influence of TnAAbs on endogenous troponin measurements was studied in 35 plasma samples from 15 patients with AMI. RESULTS The inhibitory effect of TnAAbs on the cTnI immunodetection was observed only for the ternary cardiac troponin complex (I-T-C) and not for the binary cardiac troponin complex (I-C) or free cTnI. In the same TnAAbs-containing samples, the immunodetection of cardiac troponin T (cTnT) added in the form of I-T-C (but not free cTnT) was also inhibited in the assays that used monoclonal antibodies (mAbs) specific to the 223-242 epitope. The negative effects of TnAAbs on the measurements of endogenous cTnI in AMI samples were less than on the measurements of isolated I-T-C and decreased with time after the onset of symptoms. Early AMI blood samples might contain a mixture of the I-T-C and I-C complexes with the ratio gradually changing with the progression of the disease in favor of I-C. CONCLUSIONS The investigated TnAAbs are specific to the structural epitopes formed by cTnI and cTnT molecules in the I-T-C complex. AMI blood samples contain a mixture of I-C and I-T-C complexes. The concentrations of total cTnI at the early stage of AMI could be underestimated in approximately 5%-10% of patients if measured by TnAAbs-sensitive immunoassays.

Comparison of soluble and placental transferrin receptors as standards for the determination of soluble transferrin receptor in humans

Transferrin receptor is a transmembrane protein that mediates iron transport from blood into cells. The extracellular part of this receptor circulates in blood as soluble transferrin receptor (sTfR) and the immunological determination of this parameter is widely used in clinical practice. This study aimed at comparing the properties of sTfR and placental TfR (pTfR) and to evaluate the validity of pTfR as a standard for the determination of sTfR in human serum. sTfR and pTfR were studied by immunofluorescent assay and fast protein liquid chromatography (FPLC) gel filtration. Serum sTfR levels were calculated using sTfR or pTfR as a standard. The immunological activity of pTfR was lower than that of sTfR in all anti‐TfR monoclonal antibody pairs. Upon FPLC gel filtration, pTfR eluted in a void volume of the column as a protein with a molecular weight (MW) of >1500 kDa, whereas the MW of sTfR corresponded to 237 kDa. This could be a result of micelle formation by pTfR because of its hydrophobic intracellular part. The serum sTfR levels calculated against sTfR were 2.5 times lower than those calculated against pTfR. Serum sTfR levels are overestimated when pTfR is used as the standard.

Glycosylated and non-glycosylated NT-IGFBP-4 in circulation of acute coronary syndrome patients

BACKGROUND N-terminal and C-terminal proteolytic fragments of IGF binding protein 4 (NT-IGFBP-4 and CT-IGFBP-4) were recently shown to predict adverse cardiac events in acute coronary syndrome (ACS) patients. NT-IGFBP-4 and CT-IGFBP-4 are products of the pregnancy-associated plasma protein-A (PAPP-A)-mediated cleavage of IGFBP-4. It has been demonstrated that circulating IGFBP-4 is partially glycosylated in its N-terminal region, although the influence of this glycosylation on PAPP-A-mediated proteolysis and the ratio of glycosylated/non-glycosylated IGFBP-4 fragments in human blood remain unrevealed. The aims of this study were to investigate i) the presence of glycosylated NT-IGFBP-4 in the circulation, ii) the influence of the glycosylation of IGFBP-4 on its susceptibility to PAPP-A-mediated cleavage, and iii) the influence of glycosylation on NT-IGFBP-4 immunodetection. METHODS Affinity purification was used for the extraction of IGFBP-4 and NT-IGFBP-4 from plasma samples. Purified proteins were quantified by Western blotting and specific sandwich immunoassays, while molecular masses were determined using mass spectrometry. RESULTS Glycosylated NT-IGFBP-4 was identified in the blood of ACS patients. The fraction of glycosylated NT-IGFBP-4 in individual plasma samples was 9.8%-23.5% of the total levels of NT-IGFBP-4. PAPP-A-mediated proteolysis of glycosylated IGFBP-4 was 3-4 times less efficient (p < 0.001) than proteolysis of non-glycosylated protein. A sandwich fluoroimmunoassay that was designed for quantitative NT-IGFBP-4 measurements recognized both protein forms with the same efficiency. CONCLUSIONS Although glycosylation suppresses PAPP-A-mediated IGFBP-4 cleavage, a considerable amount of glycosylated NT-IGFBP-4 is present in blood. Glycosylation does not influence NT-IGFBP-4 measurements using a specific sandwich immunoassay.