Comparison of alpha-1-acid glycoprotein isoforms from healthy and cancer patients by capillary IEF

a-1-Acid glycoprotein (AGP) is a glycoprotein that presents different forms in the same in- dividual, depending on the amino acid sequence and/or on the carbohydrate distribution of each form. Changes in these two types of heterogeneities are related to pathophysiological states. The aim of this work is to study the possibility of comparing AGP samples in terms of their CIEF profiles, what would facilitate in a future to perform studies about the role of AGP as a disease marker. In the present study, the CIEF profiles of AGP samples purified from sera of healthy donors and of ovary cancer and lymphoma patients are qualitatively and quantitatively compared. To make possible the comparison of those electrophoretical profiles, reliable assignment of AGP peaks is necessary. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Percentages of correct assignment of AGP peaks using the migration time of each peak relative to the migration time of an internal standard close to 95% are achieved. After peak assignment, a different distribution of the area percentage of AGP forms is observed when comparing samples from diseased and healthy individuals, the most acidic AGP forms being present in a higher proportion in the samples from cancer patients. Although the number of samples studied is too low to get any clinical significance from these results, this work provides a way to study the role of AGP as a biomarker.Peer reviewe

Increasing DNA isolation performance from blood cells using a modified commercial low-cost reagent

Background: Genomic studies facilitate comprehension of the pathophysiology, diagnosis, and treatment of chronic diseases. Such studies require sufficient and good quality DNA isolated from a large number of blood samples. This study attempts to obtain a high-quality genomic DNA isolated from a large number of blood samples using a simple and cheap method. Methods: The EasyPure® Genomic DNA Kit (Transgen Biotech) was modified to increase the amount of DNA recovery: a few steps and two additional column elutions were added to the original manufacturer´s procedure. Results: The amount of DNA isolated from frozen blood samples increased by an average of 56%. Its 260/280 ratio and electrophoretic mobility properties make it suitable for genomic studies. Conclusions: A relatively low-cost commercial column and a simple modification of the manufacturer´s protocol, provided a simple and cheap procedure to isolate high-quality DNA from a large number of blood samples suitable for genomic studies.Fil: Kraemer, Mauricio Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Endocrinología Experimental y Aplicada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Endocrinología Experimental y Aplicada; ArgentinaFil: Mencucci, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Endocrinología Experimental y Aplicada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Endocrinología Experimental y Aplicada; ArgentinaFil: Abba, Martín Carlos. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lacunza, Ezequiel. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gagliardino, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Endocrinología Experimental y Aplicada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Endocrinología Experimental y Aplicada; Argentin

Comparison of alpha-1-acid glycoprotein isoforms from healthy and cancer patients by capillary IEF

a-1-Acid glycoprotein (AGP) is a glycoprotein that presents different forms in the same in- dividual, depending on the amino acid sequence and/or on the carbohydrate distribution of each form. Changes in these two types of heterogeneities are related to pathophysiological states. The aim of this work is to study the possibility of comparing AGP samples in terms of their CIEF profiles, what would facilitate in a future to perform studies about the role of AGP as a disease marker. In the present study, the CIEF profiles of AGP samples purified from sera of healthy donors and of ovary cancer and lymphoma patients are qualitatively and quantitatively compared. To make possible the comparison of those electrophoretical profiles, reliable assignment of AGP peaks is necessary. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Percentages of correct assignment of AGP peaks using the migration time of each peak relative to the migration time of an internal standard close to 95% are achieved. After peak assignment, a different distribution of the area percentage of AGP forms is observed when comparing samples from diseased and healthy individuals, the most acidic AGP forms being present in a higher proportion in the samples from cancer patients. Although the number of samples studied is too low to get any clinical significance from these results, this work provides a way to study the role of AGP as a biomarker.Peer reviewe

Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or car- diovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time-and labour-consuming, and generally they have not been proven to be compatible with capil- lary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interfer- ents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chro- matography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.Sara Ongay and Izaskun Lacunza acknowledge CSIC for contracts to perform Ph.D. Theses. Drs. Enrique Caso and Maria del Val Toledo are acknowledged for providing the serum samples. Financial sup- port from the Spanish Ministry of Science and Innovation (projects CTQ 2006-05214, HH 2006-0013, and PSE-010000-2008-6) and Comunidad de Madrid (project S-GEN-0247-2006) is acknowledged.Peer reviewe

Immunochromatographic removal of albumin in erythropoietin biopharmaceutical formulations for its analysis by capillary electrophoresis

Human serum albumin (HSA) is added to some pharmaceutical preparations as an excipient. This is the case for some of the commercial preparations of recombinant erythropoietin (rEPO). Differences in the number of the sialic acid moieties in the different rEPO glycoforms confer to these forms different net charges and different bioactivity. Knowledge of the isoforms present in each pharmaceutical product is then of interest. Differences in net charge of the rEPO forms make possible their separation by electrophoretical methods. However it has been observed in our laboratory that the amount of HSA usually present in these drug formulations interferes or even precludes separation of rEPO bands by capillary zone electrophoresis (CZE). In this work, an immunochromatographic method to remove HSA from rEPO biopharmaceutical formulations and a procedure to concentrate the sample that is needed to be performed prior to the analysis by CZE are developed. A home-made computer program to compare the percentage of correct assignments of electrophoretical bands provided by different migration parameters is used to study the effect of HSA remaining in samples on the accuracy of assignment of rEPO bands. When there exists a residual concentration of HSA in the sample (<2 mg/ml) only the effective electrophoretic mobility is a reliable migration parameter to assign rEPO bands with a 100% of correct assignment. This parameter allows the correct assignment of bands of rEPO from pharmaceutical products formulated with HSA after immunochromatographic removal of HSA. Electrophoretical bands found in epoetin α, one of the commercial formulations of rEPO, are independent of the molecular mass of the excipients. The methodology used in this work for the analysis by CZE and the assignment of rEPO isoforms, as well as for the immunochromatographic HSA removal in the pharmaceutical products could be of high interest for the health authorities to control the quality of the product in marketing surveillance studies and for the quality control laboratories of the manufacturers.The authors are grateful to foundation R. Areces and to Spanish Ministry of Science and Technology (project TIC2003- 01906) for providing financial support, to European Pharma- copoeia for providing the rEPO BRP used as standard sample, to Dr. Michel Girard (Health Canada) for the gift of epoetin alfa;, and to Applied Biosystems for the immunocolumn kindly donated. P. L-Q. and I. L. acknowledge Spanish Ministry of Education and Science and C. S. I. C., respectively, for fellowships.Peer reviewe

Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis

Capillary zone electrophoresis of samples of recombinant human erythropoietin is per- formed. An in-house computer program is developed to compare the reliability of different migration parameters to assign the close migration bands of isoforms of erythropoietin. The migration time relative to the electroosmotic flow marker and the effective electrophoretic mobility are selected as the most accurate parameters. Percentages of correct assignment of bands higher than 99% are obtained with these parameters even when changes in operational factors are introduced. The chosen parameters have been applied to assign bands of isoforms in commercial samples of a-and b-epoetin. The same capillary electrophoresis method has been applied to separate bands of isoforms of an erythropoietin analogue, darbepoetin a, the novel erythropoiesis-stimulating protein.Peer reviewe

Noncardiac Production of Soluble ST2 in ST-Segment Elevation Myocardial Infarction

This work has been supported by two grants (PI14/01637, PI17/01742) from “Instituto de Salud Carlos III”, Madrid, Spain, and a grant (19334/PI/14) from “Fundación Séneca de Ciencia y Tecnología de la Región de Murcia”, Murcia, Spain

Circulating microRNAs as biomarkers of Chagas cardiomyopathy

Background: Chagas cardiomyopathy (CHCM) is the most important clinical manifestation of Chagas disease. The analysis of cardiac miRNAs may contribute to predicting the progression to CHCM in Chagas indeterminate phase and/or to the differential diagnosis for cardiomyopathy. Methods: We carried out a case-control study to identify circulating miRNAs associated with CHCM. We assigned 104 participants to four groups: healthy controls (HC), Chagas non-cardiomyopathy controls, CHCM cases, and ischemic cardiomyopathy controls. We performed a clinical, echocardiographic, and laboratory evaluation and profiled circulating miRNA in the serum samples. Results: Differences between groups were observed in clinical variables and in the analysis of miRNAs. Compared to HC, CHCM participants had 4 over-expressed and 6 under-expressed miRNAs; miR-95-3p and miR-130b-3p were upregulated in CHCM compared with controls, Chagas non-cardiomyopathy and ischemic cardiomyopathy participants, suggesting that might be a hallmark of CHCM. Analysis of gene targets associated with cardiac injury yielded results of genes involved in arrhythmia generation, cardiomegaly, and hypertrophy. Conclusions: Our data suggest that the expression of circulating miRNAs identified by deep sequencing in CHCM could be associated with different cardiac phenotypes in CHCM subjects, compared with Chagas non-CHCM, ischemic cardiomyopathy controls, and healthy controls.Fil: Antonietti, Laura. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; Argentina. Universidad Nacional Arturo Jauretche; ArgentinaFil: Mariani, Javier. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; Argentina. Universidad Nacional Arturo Jauretche; ArgentinaFil: Martínez, María Jose. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Santalla, Manuela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Vensentini, Natalia. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Kyle, Diego Alfredo. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; Argentina. Universidad Nacional Arturo Jauretche; ArgentinaFil: de Abreu, Maximiliano. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; Argentina. Universidad Nacional Arturo Jauretche; ArgentinaFil: Tajer, Carlos Daniel. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; Argentina. Universidad Nacional Arturo Jauretche; ArgentinaFil: Lacunza, Ezequiel. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferrero, Paola Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentin