ABSTRACT
The atrial natriuretic peptide (ANP) and its precursor (N-terminal fragment of atrial natriuretic peptide, NT-proANP) are natriuretic peptides released into the circulation as a consequence of an acute atrial stretch. As for the brain natriuretic peptide and its N-terminal fragment, the biological significance of ANP and NT-proANP has been widely studied in humans, but the literature is lacking information about the determination of these biomarkers in veterinary medicine and, in particular, in the toxicological species used in preclinical pharmaceutical drug development. This paper describes the evaluation of ANP and NT-proANP levels in a healthy population of Han Wistar and Sprague-Dawley rats, as well as in a rodent model of hypertension (Spontaneously Hypertensive rats). Both biomarkers were measured by mean of two commercially available enzyme immunoassays and serum levels were correlated with heart weight and histopathological findings in the heart, with the aim of building an integrated assessment of the significance of these biomarkers. Results obtained demonstrated that NT-proANP and ANP can be accurately measured in the different rat strains, with NT-proANP concentrations higher than those of ANP, as expected because of its longer half-life. In addition, both correlated well with cardiac hypertrophy evaluated by means of heart weight and histopathological examination. NT-proANP and ANP represent reliable markers of cardiac hypertrophy in the rat.
Subject(s)
Atrial Natriuretic Factor/blood , Biomarkers/blood , Animals , Blood Pressure , Cardiomegaly/blood , Half-Life , Heart Atria/metabolism , Male , Organ Size , Radioimmunoassay , Rats , Rats, Inbred SHR , Rats, Sprague-Dawley , Rats, WistarABSTRACT
BACKGROUND: Serum protein analysis in both humans and experimental animal species has so far been carried out by labor-intensive techniques, such as agarose gel electrophoresis (AGE). OBJECTIVE: The objective of this study was to evaluate capillary electrophoresis (CE) as an alternative technique to AGE for the analysis of serum proteins from healthy animals. METHODS: Blood samples were collected into tubes without anticoagulant from 6 fasted healthy male mice, rats, dogs, marmosets, and humans. Serum proteins were separated by CE using a technique standardized for the analysis of human proteins, and the results (efficiency, resolution, and precision) were compared with those obtained through AGE. RESULTS: Compared with AGE, CE resulted in narrower peaks and more peaks. The efficiency of protein separation by CE was significantly higher for all species, and resolution (R) was significantly higher in samples from dogs. Using rat serum, intraday reproducibility was lower for all protein fractions, and interday reproducibility was lower for most peaks, compared with AGE. CONCLUSIONS: We conclude that CE is a viable alternative to AGE for the determination of protein electrophoresis in a routine veterinary clinical pathology laboratory. The minimal sample requirement (2 microL), complete automation, and quantitative results make CE an especially valuable technique for protein analysis in experimental animal models.
Subject(s)
Blood Proteins/analysis , Callithrix/blood , Electrophoresis, Agar Gel/methods , Electrophoresis, Capillary/methods , Animals , Dogs , Humans , Mice , RatsABSTRACT
This work represents a first attempt to refine the colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic assay by incorporating liver microsomes and co-factors as a metabolic system into the in vitro test system in response to an ECVAM recommendation. From the comparison of results obtained with the CFU-GM clonogenic assay currently used and with the new experimental protocol, different toxicity on granulocyte/macrophage precursors was demonstrated, when drugs with a known metabolism in vivo were tested.
Subject(s)
Colony-Forming Units Assay/methods , Granulocytes/drug effects , Hematopoietic Stem Cells/drug effects , Macrophages/drug effects , Microsomes, Liver/metabolism , Animals , Cefazolin/toxicity , Cefotaxime/toxicity , Ciprofloxacin/toxicity , Doxorubicin/toxicity , Granulocytes/physiology , Macrophages/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Rats , Rats, Sprague-Dawley , Valproic Acid/toxicity , Zidovudine/toxicityABSTRACT
In this work, a first attempt to set-up a new in vitro experimental protocol with culture in liquid medium and flow cytometry analysis of bone marrow progenitors is described. This protocol is proposed as an alternative to the colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic in vitro assay currently used to assess the toxic potential of new drugs in the bone marrow. This new experimental approach should enable to speed up the procedure of the in vitro haematotoxic potential assessment, to reduce inter-experimental variability and to enhance result accuracy. Preliminary results obtained demonstrated that the progenitor cell count by flow cytometry replacing the light microscopy granulocyte/macrophage colony count represents a tremendous improvement in terms of accuracy and standardisation. Moreover, differential counts of cell sub-populations can be performed by using specific monoclonal antibodies. Furthermore, this method demonstrated to be time-saving, since 4 day cell incubation period is required instead of 7-14 day incubation in the CFU-GM clonogenic assay. On the basis of results obtained so far, the new experimental protocol proposed looks a promising alternative to the CFU-GM clonogenic assay currently used.
