Browsing by Subject "Plasma"
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- PublicationOpen AccessA Fast and Accurate Method for the Quantification of Doxycycline in Goat Plasma and Milk by HPLC Using a Fluorescence Detector(2024-12-17) Martínez, José; Hernandis, Verónica; Badillo, Elena; Escudero, Elisa; Yuste Pérez, María Teresa; Galecio, Juan Sebastián; Marín, Pedro; FarmacologíaDoxycycline is an antimicrobial agent used in veterinary medicine to treat a variety of bacterial infections. To date, no analytical technique utilising HPLC with fl uorescence detection has been documented for the quantifi cation of doxycycline concentrations in goat plasma or milk. Consequently, the objective of the present study was to propose a rapid HPLC assay with fl uorescence detection for the quantifi cation of doxycycline in the aforementioned samples, thereby facilitating the conduct of pharmacokinetic studies and the detection of residues in diverse goat tissues. Proteins were precipitated with methanol and trifl uoroacetic acid in a single step. Doxycycline was separated on a XBRIDGE C18 column using an isocratic method. Sample volume injected into the HPLC system was 50 µl. Fluorescence detection was conducted with an excitation wavelength of 380 nm and an emission wavelength of 520 nm. The retention times of doxycycline and danofl oxacin (internal standard) were determined to be 8.0 and 5.5 minutes, respectively. The calibration curves for plasma and milk exhibited linearity over the concentration range of 0.1 to 2 μg/mL. The limit of detection was 0.065 μg/ mL, while the limit of quantifi cation was 0.1 μg/mL in both matrices. The accuracy and precision of the method were consistently within the limits of 10.9% for plasma and 10.5% for milk. The fi ndings of this study may be employed in the quantifi cation of doxycycline in goat plasma and milk, thus facilitating the conduct of pharmacokinetic studies.
- PublicationOpen AccessAmelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism(American Society of Hematology, 2012-04-12) Martínez-Martínez, Irene; Navarro-Fernández, José; Ostergaad, Alice; Gutierrez-Gallego, Ricardo; Padilla, José; Miñano, Antonia; Pascual, Cristina; Martínez, Constantino; Morena-Barrio, María Eugenia de la; Pedersen, Shona; Kristensen, Soren Risom; Corral, Javier; Bohdan, Nataliya; Morena Barrio, María Eugenia de la; Águila Martínez, Sonia; Vicente García, Vicente; Corral de la Calle, Javier; MedicinaThe balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the β-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of α-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin
- PublicationOpen AccessDiagnostic value of matrix metalloproteinase 9 and tissue inhibitor of matrix metalloproteinases 1 in cholesteatoma(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Olszewska, Ewa; Matulka, Marlena; Mroczko, Barbara; Pryczynicz, AnnaObjectives: Matrix metalloproteinase 9 (MMP-9), able to degrade type IV collagen, plays a key role in inflammatory cell migration as well as in the destructive behaviour of cholesteatoma. The aim of our study was to compare the expression of MMP-9 and TIMP-1 in cholesteatoma tissue and in the concentrations in serum and plasma concentrations. Material and Methods: Twenty five adult patients suffering from cholesteatoma (a study group) were included in the study. A comparison group consisted of 25 adult patients admitted to hospital due to nasal septum deviation. MM-9 and TIMP-1 serum and plasma concentrations as well as proteins’ expressions in cholesteatoma tissues (study group) and normal retroauricular skin specimens (control group) were evaluated. MMP-9 and TIMP-1 concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Cholesteatoma tissues and normal retroauricular skin specimens were evaluated immunohistochemically. Results: In the study and comparison groups, MMP9 and TIMP-1 concentrations were similar with no significant difference within the groups. In cholesteatoma tissues, the expression of the investigated enzyme and its inhibitor was higher than in normal skin specimens, limited mostly to cholesteatoma perimatrix. Conclusion: Cholesteatoma may be limited to the middle ear or parts of the temporal bones. Our findings suggest better clinical usefulness of MMP-9 and TIMP-1 expression in cholesteatoma tissues than either serum or plasma levels of these proteins. It might suggest that the higher the expression of MMP-9 the stronger the inflammation -accompanied cholesteatoma.
- PublicationOpen AccessEffect of regenerative factor rich plasma, P substance and fetal calf serum on the growth of epithelial cells in the cornea. Comparative experimental study(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2013) Márquez de Aracena del Cid, R.; Pérez Ordoñez, A.Purpose. The goal of this study was to evaluate the experimental effectiveness of Regenerative Factor Rich Plasma (RFRP) of human blood versus Fetal Bovim Serum (FBS) and neuropeptide Substance P (SP) on corneal epithelium cell proliferation. Method. Rabbit corneal epithelium cell (CCL-60) growth was compared between different RFRP fractions, FBS and with the neuropeptide Substance P. The ability of the RFRP fractions and SP to revert the inhibitory effect of the CsA was also evaluated. Results. All groups showed an increase (p<0.001) in corneal epithelial cell growth compared with the control group. The maximum capacity of cell growth was obtained with dilutions of 50% in the FBS, RFRP-I, RFRP -II, RFRP-III groups and with 100nM of SP. The highest growth was observed with 50% FBS, RFRP-I and RFRP-II. The group with SP and RFRP-III had significantly lower growth (p<0.001). When the NK1 receptor antagonist CsA was added at a dose of IC50, we found a significant decrease in cell growth (p<0.001) in all culture conditions, including the control group. The decrease was similar in all groups, but was especially pronounced in RFRP-II. RFRP I, II and III promoted growth more than SFB 10%. Conclusion. The RFRP of human blood promotes the growth of corneal epithelial cells in a significantly more efficient manner than FSB and SP. RFRP can be effective both in cell cultures and stem cell cultures.
- PublicationRestrictedTrace elements in saliva and plasma of patients with type 2 diabetes: Association to metabolic control and complications(Elsevier Ltd., 2019-10-08) Marin Martinez, Luis; Molino Pagan, Diana; Lopez Jornet, Pia; Dermatología, Estomatología, Radiología y Medicina FísicaAim: An analysis is made of the saliva and plasma levels of trace elements in patients with type 2 diabetes mellitus and their association to metabolic control and the presence of chronic complications. Methods: A cross-sectional observational clinical study was carried out in 74 patients with type 2 diabetes mellitus. Mass spectrometry (ICP-MS) was used to determine the following trace elements in plasma and unstimulated basal saliva: 13Al, 16S, 4Be, 5B, 20Ca, 27Co, 29Cu, 24Cr, 38Sr, 15P, 3Li, 12Mg, 25Mn, 28Ni, 82Pb, 37Rb, 22Ti, 23V and 30Zn. Results: The levels of cobalt (p = 0.048) in saliva and of strontium (p = 0.001) in plasma were related to the presence of chronic complications. Significant differences with respect to metabolic control were observed for beryllium (p = 0.038), boron (p = 0.023) and phosphorus in saliva (p = 0.046), and for rubidium (p = 0.005), titanium (p = 0.016) and zinc in plasma (p = 0.013). A significant correlation (p < 0.001) was found between boron in plasma and boron in unstimulated basal saliva. Conclusions: The determination of trace elements in plasma and saliva constitutes a complementary tool for the assessment of metabolic control and for predicting chronic complications associated to type 2 diabetes mellitus. Further studies involving the biomonitoring of trace elements in saliva and plasma are needed
- PublicationOpen AccessTumor DNA circulating in the plasma might play a role in metastasis. The hypothesis of the genometastasis(Murcia : F. Hernández, 1999) García-Olmo, Dolores C.; Ontañon, J.; Martinez, E.Background: Clinical and experimental observations suggest that more than one pathway might be involved in the development of metastases. In the present study, we examined the presence of tumor DNA in plasma using an experimental model in which tumor cells were modified with a genome-associated tag. We also investigated whether plasma of tumor-bearing rats had any effect on cultured cells and healthy animals. Methods: Transfected cancer cells (DHDlK12-PROb stably transfected with pCDNA3.1CAT.) were injected subcutaneously into the chest of BD-IX rats. Animals were divided into ten groups according to the time between injection of tumor cells and euthanasia. Prior to euthanasia (2-14 week), blood samples were collected by cardiac puncture. To detect circulating tumor cells and CAT-encoding DNA in plasma, we performed PCR with nested primers. Fifty samples of plasma were chosen at random to supplement the medium of fifty cultures of DHD cells for 10-12 days. PCR for the detection of CAT DNA in cells was performed approximately one to two months later. Four healthy rats received an intraperitoneal injection of plasma from a tumor-bearing rat five times at week for 4 to 6 weeks. Animals were sacrificed and samples of liver, kidney, spleen, omentum, blood and lung were processed by PCR for the detection of CAT DNA. Results: Detection of CAT DNA in plasma was slightly more frequent than in the buffy-coat fraction. All surviving cultures that had been supplemented with plasma were positive at some point for CAT DNA. In all four healthy animals injected with plasma of tumor-bearing rats, the marker gene for CAT was found in extracts of lungs. Conclusion: Our present observation lead us to propose the following hypothesis. Metastases might develop as a result of transfection of susceptible cells in distant target organs with dominant oncogenes that are present in the circulating plasma and are derived from the primary tumor.