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Biomedical subjects

J L Navarro

Publications and source records attributed to J L Navarro.

At least 19 recordsLinked to original sources

Amino acid analysis by high-performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate.

Amino acids were determined by precolumn derivatization with diethyl ethoxymethylenemalonate and reversed-phase high-performance liquid chromatography (HPLC) with spectrophotometric detection at 280 nm. The reaction time was 50 min and the derivatives were stable at room temperature. Chromatographic resolution of a mixture of the derivatives of seventeen amino acids, including proline and cystine, was achieved within 35 min using a binary gradient system. The detection limit was 3 pmol. Amino acid analyses of acid hydrolysates of two proteins gave results equivalent to those obtained by conventional ion-exchange-based amino acid analysis. The simplicity of the procedure allows its use on any multi-purpose HPLC system.

Amino Acids

Expression of phosphatidylinositol anchored membrane proteins in paroxysmal nocturnal haemoglobinuria after bone marrow transplantation.

A 20-year-old male with severe bone marrow failure associated with paroxysmal nocturnal haemoglobinuria (PNH) underwent an allogeneic bone marrow transplantation (BMT). Flow cytometric analysis of phosphatidylinositol (PI) anchored membrane proteins prior to BMT showed a markedly reduced expression of monocyte CD14 and neutrophil CD16 molecules. On day +17 after BMT expression of both antigens reached normal values and remained stable throughout a follow-up period of 10 months, thus confirming the eradication of the PNH clone. To date, this is the first case in which normal expression of PI-anchored proteins after BMT is reported.

Adult

Hereditary hemorrhagic telangiectasia: analysis of platelet aggregation and fibrinolytic system in seven patients.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder characterized by the presence of generalized mucocutaneous and visceral telangiectasias associated with recurrent bleeding. In order to analyze the mechanism of bleeding in HHT we studied the hemostatic system and platelet in vitro aggregation in 7 unrelated patients suffering from HHT. Unlike the authors of previous reports, we could not find any significant alteration of the parameters measured suggesting a possible local role of the affected endothelial cells.

Blood Proteins

[Hemostasis profiles in thrombotic disease].

It is well known that in thrombotic disease the alteration of biological factors such as antithrombin III, protein C, and protein S deficiency, and congenital disfibrinogenimias and displasminogenemias are determining factors being the acquired alterations not so well known. With this in mind was studied 85 patients with arterial thrombosis and 196 with venous thrombosis, who were again divided into three groups: unique or of repetition, less or more than 35 years and with or without immediate apparent cause. The general clinical-biological profile in patients with thrombosis in whom a congenital deficit is not detected, can help establish prognosis and treatment in these patients. In our patients, together with the importance of factors such as obesity, hyperlipemia, and tabaquism, an increase in fibrinogen (Fg), antigenic Factor VII (vWF:Ag), total protein S is observed as well as a decrease in total fibrinolytic activity related to an increase in the inhibitor of the plasminogen tissue activator (PTA).

Adolescent

Acute lymphoblastic leukemia in an XYY male.

A case of acute lymphoblastic leukemia (ALL) in a 16-year-old male with a 47,XYY karyotype is reported. This chromosome aneuploidy was found in both bone marrow (BM) cells and mitogen-stimulated lymphocytes. Immunologic profile of leukemic cells showed a null phenotype. To our knowledge, this is the fifth case reported in the literature.

Adolescent

The fine structural localization of endogenous and exogenous peroxidase activity in human bone marrow mast cells under pathological conditions.

We have examined the ultrastructural characteristics of peroxidase activity in human bone marrow mast cells. These studies were performed in three patients with systemic mast cell disease, and in another six patients showing bone marrow mast cell hyperplasia. Endogenous peroxidase activity was localized in the perinuclear cisternae and strands of endoplasmic reticulum, but never in the granules. We have also demonstrated the "in vivo" existence of exogenous peroxidase activity in two of the three cases of systemic mast cell disease. The peroxidase internalization involved its binding to the plasma membrane, followed by its incorporation into the cell by a general endocytic process comprising the uptake of dispersed peroxidase-positive material mainly by phagocytosis of granular structures containing peroxidase. The exogenous peroxidase appeared in non-membrane bound granules, vacuoles or aggregates, but we have never seen the enzyme linked to the mast cell granules.

Adult

Arachidonic acid metabolism in platelets stored for 5 days.

(C-14)-Arachidonic acid [(C-14)-AA] metabolism was studied in platelet concentrates (PCs) stored for 5 d. There was a gradual decrease in uptake of radioactivity from day 0 to 3 (P less than 0.01). On day 0, distribution of radioactivity in platelet phospholipids (PLs), and formation of phosphatidic acid, HETE and cyclooxygenase products, when platelets were exposed to thrombin (5 U/ml), were similar to that reported for fresh platelets. On day 3 there was a change in the distribution of (C-14)-AA in platelet PLs which consisted of an increase in the percentage of radioactivity bound to phosphatidylserine, from 5.3 +/- 0.9% on day 0 to 8.8 +/- 1.5% on day 3 (P less than 0.001), and a decrease in (C-14)-AA in phosphatidylinositol (PI), from 12.4 +/- 1.5% on day 0, to 7.9 +/- 0.9% on day 3 (P less than 0.001). Phosphatidic acid generated by thrombin-stimulated platelets on day 0, comprised 2.6 +/- 0.5% of total radioactivity, but dropped to 1.4 +/- 0.3% on day 3 (P less than 0.001), and 0.9 +/- 0.2% on day 5 (P less than 0.01). These values showed a good correlation with the percentage of (C-14)-AA released from PI on the same days (r = 0.9). On day 0, 13.4 +/- 4.4% of platelet radioactivity was released from phosphatidylcholine by thrombin, but this amount was reduced to 6.8 +/- 3.4% on day 5 (P less than 0.05). Generation of radioactive 12-hydroxy-5,8,10,14-eicosatetraynoic acid (HETE) also dropped from 7.2 +/- 2.9 on day 0, to 2.1 +/- 1 on day 5 (P less than 0.01). We could not detect changes in cyclooxygenase metabolites. In conclusion, we suggest that various enzymatic pathways implicated in AA metabolism by platelets are impaired by storage.

Arachidonic Acid

Thrombus age and tissue plasminogen activator mediated thrombolysis in rats.

Thrombolytic efficacy is directly related to thrombus age. We used recombinant tissue plasminogen activator (rt-PA), Streptokinase (SK) and Urokinase (UK) on a seven days old inferior vena cava thrombus model. "In vitro" clot lysis assays with fibrinogen-I125 were also evaluated with the same agents at 1, 3 and 7 days. Fibrinogen, D-D dimer and t-PA were measured. Experiments with 40 controls and 27 rt-PA treated animals showed a significant decrease in thrombus weight (8.5 +/- 1.1 mg) vs. (4.2 +/- 0.6 mg) (p less than 0.01). Fibrinogen concentration in rt-PA group decreased significantly (1032 +/- 123 mg/dl) vs. (202 +/- 32 mg/dl) (p less than 0.001). "In vitro" rt-PA showed a marked lytic effect in a wide range (100-4 IU/ml). Fibrin selective agents as rt-PA may be more effective than non selective ones in the treatment of fully developed thrombus.

Animals

[Thrombolytic agents: efficacy in relation to evolution time and dose].

"In vitro" efficacy of urokinase (UK), streptokinase (SK) and tissue-type plasminogen activator (t-PA) was studied. Lytic action was measured by dry clot weight, 125I counts released from previously incorporated 125I-fibrinogen and fibrin fragment quantification at 1, 3 and 7 days using different doses. t-PA doses between 100 and 0.8 IU/ml were efficient. t-PA (40 IU/ml), SK (200 IU/ml) and UK (200 IU/ml) showed an equivalent activity in 1, 3 and 7 day-old clots. Fibrin fragment (D-D dimer) production was highest when t-PA and UK were used. "In vitro" lytic capacity up to 7 days after clot formation has been proved with the different fibrinolytic agents.

Blood Coagulation

[The modified "pink test" in the diagnosis of hereditary spherocytosis].

Hereditary spherocytosis is still difficult to diagnose in some situations due to the existence of subclinical expressions and the lack of an accurate test with high sensitivity and specificity. Recently, Vettore et al. described the so-called 'Pink test' as an easy method with the highest reproducibility and sensitivity. Herein, we present our experience with the 'Pink test' in the diagnosis of 16 previously diagnosed hereditary spherocytosis patients comparing the results with those obtained in 96 healthy controls, 41 beta-thalassaemias, 9 autoimmune haemolytic anaemias, 8 chronic hemoproliferative syndromes and 2 patients with pyruvate kinase deficiency. We also present a modification of the original 'Pink test' in which a small sample of blood (200 microL) obtained by finger-prick (or heel puncture in newborns and infants) is mixed with the hemolyzing solution of the 'Pink test' within the first three hours after blood drawing. Elevated correlation coefficients (r = 0.75-0.96) between both methods have been obtained comparing the percentage of final haemolysis in 25 healthy controls, 21 beta-thalassaemia minor and 9 hereditary spherocytosis patients.

Adult

[Measurement of phospholipids in erythrocytes, plasma and platelets in a patient with hereditary spherocytosis].

The amount and distribution of phospholipids (PLs) were studied in erythrocytes, plasma and platelets of one patient with hereditary spherocytosis (HS). Phosphatidylethanolamine (PE) was always decreased. Total PLs amount was remarkably reduced in erythrocytes but normal in platelets. Since platelets do not contain those proteins which have been implicated in the red cell abnormalities of patients with HS, our findings reinforce the theory that a defective protein-lipid interaction causes the abnormal lipid loss in the erythrocytes of these patients.

Adult

C18 solid-phase isolation and high-performance liquid chromatography/ultraviolet diode array determination of fully methoxylated flavones in citrus juices.

A new analytical methodology for the determination of fully methoxylated flavones (FMFs) in citrus juices is described. Isolation of the FMFs is carried out by percolation of 30 mL of clarified citrus juice (to which tetramethyl-o-kaempferol is previously added as internal standard) through a C18 Sep-Pak cartridge, washing with 3 mL of water followed by 5 mL of water/acetonitrile (3:1), and selective elution of the retained FMFs with 5 mL of water/acetonitrile (9:11). Determination of the isolated FMFs is carried out by reversed-phase high-performance liquid chromatography (HPLC) and UV diode array detection (DAD). Signals at wavelengths 320, 335, and 345 nm (bandwidth 4 nm) are simultaneously acquired, stored, plotted, and integrated. The column used is a microbore (200 x 2.1-mm) Hypersil ODS 5 microns. Elution is in gradient mode, using a ternary mobile phase (water/acetonitrile/tetrahydrofuran). Column temperature is 40 degrees C. Recovery yields are nearly 100% for all the FMFs detected and identified: isosinensetin, hexamethyl-o-gossypetin, sinensetin, tetramethyl-o-isoscutellarein, hexamethyl-o-quercetagetin, nobiletin, tetramethyl-o-scutellarein, heptamethoxyflavone, and tangeretin. Chromatographic separation of the FMFs is extremely dependent upon the minor changes of the mobile phase composition and percentages, gradient rate, and temperature. The UV spectra (230 to 400 nm) of the FMFs obtained under chromatographic conditions are given. The FMFs relative response factors at 320, 335, and 345 nm and their concentrations in hand-squeezed and commercial concentrated orange and mandarin juices are tabulated. The FMF concentration differences found among samples are discussed.

Beverages

Human bone marrow mast cells in systemic mast cell disease. Ultrastructure and evaluation of fixation and staining techniques.

In a case of systemic mast cell disease with moderate bone marrow involvement, we studied the sensitivity of mast cell cationic dye-binding to formaldehyde fixation, as well as the mast cell ultrastructure, so as to determine whether these cells possess characteristics of mucosal mast cells. This histochemical method has proved to be one of the few which reveal heterogeneity of the mast cells in human intestine. Even though rat bone marrow mast cells have been shown to belong to the mucosal mast cell compartment, we have found no difference in mast cell counts in samples fixed either in formaldehyde or Carnoy's fixative. The ultrastructure did not show major differences with cutaneous mast cells. A few cells presented two nuclei, suggesting mitotic division of mature mast cells in bone marrow.

Adult