A randomized clinical trial to determine the effect of angiotensin inhibitors reduction on creatinine clearance and haemoglobin in heart failure patients with chronic kidney disease and anaemia.

BACKGROUND: Chronic kidney disease is a common comorbidity in elderly patients with heart failure. Evidence supports the use of angiotensin inhibitors for patients with heart failure. However, there is little evidence with which to assess the risk and benefits of this treatment in elderly patients with renal dysfunction. OBJECTIVE: To determine the efficacy and safety of angiotensin inhibitor reduction in patients with heart failure, chronic kidney disease and anaemia. STUDY DESIGN: Open randomized controlled clinical trial. SETTING: Complexo Hospitalario Universitario A Coruña (Spain). PATIENTS: Patients ≥ 50 years old, with heart failure, haemoglobin (Hb) < 12 mg/dl and creatinine clearance <60 ml/min/1.73 m(2) admitted to hospital, in treatment with angiotensin inhibitors. Informed consent and Ethical Review Board approval were obtained. INTERVENTION: A 50% reduction of angiotensin inhibitor dose of the basal treatment on admission (n = 30) in the intervention group. Control group (n = 16) with the standard basal dose. MAIN OUTCOME MEASURE: Primary outcome was difference in Hb (gr/dl), creatinine clearance (ml/min/1.73 m(2) ) and protein C (mg/dl) between admission and 1-3 months after discharge. Secondary outcome was survival at 6-12 months after discharge. RESULTS: Patients in the intervention group experienced an improvement in Hb (10.62-11.47 g/dl), creatinine clearance (32.5 ml/min/1.73 m(2) to 42.9 ml/min/1.73 m(2) ), and a decrease in creatinine levels (1.98-1.68 mg/dl) and protein C (3.23 mg/dl to 1.37 mg/dl). There were no significant differences in these variables in the control group. Survival at 6 and 12 months in the intervention and control group was 86.7% vs. 75% and 69.3% vs. 50%, respectively. CONCLUSION: The reduction of the dose of angiotensin inhibitors in the intervention group resulted in an improvement in anaemia and kidney function, decreased protein C and an increased survival rate. TRIAL REGISTRATION: EudraCT: 2008-008480-10.

[Nosocomial bacteremia in the adult patient. Study of associated costs]. / Bacteriemia nosocomial en el paciente adulto. Estudio de costes asociados.

OBJECTIVE: Nosocomial infection causes a prolonged hospital stay and an increase in care costs. The objective of this study was to determine the length of stay excess and costs attributable to nosocomial bacteremia. PATIENTS AND METHODS: Retrospective study of clinical records of 148 patients with nosocomial bacteremia during 1996. A matched case-control study was performed. For matching, the following parameters were used: RDG, year of admission, age 10 years, main diagnosis and number of secondary diagnoses. Costs were determined by excess length of hospital stay and calculating alternative costs. RESULTS: Matching was obtained for 100 cases (67.5%) and cost estimation was performed. Compared with cases, non-matched cases showed differences regarding significant issues for cost, such as hospital stay ( p = 0.01), number of empirical (p = 0.001) or definitive antibiotics (p = 0.03). The median hospital stay for cases was longer than for controls (35 vs 15.5 days, respectively; p = 0.000). When only survivor case-control pairs were considered (n = 75), cases remained in hospital for a median of 36 vs 15 days for controls (p = 0.000). Hospital stay days attributable to nosocomial bacteremia were 19.5 for all matched and 21 for matched survivor cases. Only 76% of cases had stay days attributable to bacteremia. Significant differences between cases and controls included: the mean total costs of admission (p = 0.000), cost of stay (p = 0.001), pharmaceutical expenses (p = 0.000), and cost of microbiological studies (p = 0.000), laboratory work-up (p = 0.001) and radiological studies (p = 0.000). Hospital stay represented more than 60% of costs, followed by pharmaceutical expenses. Cost differences between bacteremic patients and controls, calculated in function of stay median, was 4.424 euros (p = 0.000) and 4.744 euros (p = 0.000) for alternative costs. Ten cases showed a difference that represented more than half of the total difference. CONCLUSIONS: Nosocomial bacteremia represent a stay prolongation and a significant economical burden. Hospital stay and pharmaceutical expenses accounted for the most part of the associated costs. The differences in costs obtained with both methods were small. Since not all selected cases were matched, there may be an error in the appreciation of the difference between cases and controls.

Phytic acid as an efficient low-molecular-mass displacer for anion-exchange displacement chromatography of proteins.

Phytic acid, inositol-hexaphosphoric acid, molecular mass 650, a low-molecular-mass compound, has been identified as a nearly ideal displacer in anion-exchange displacement chromatography for the concentration and purification of model protein mixture. The concentration of low-molecular-mass displacer is a very important parameter for successful separation by displacement chromatography. Displacer concentration influences the formation of the isotachic train and the yield and recovery of the displacement chromatographic process. There is an optimum displacer concentration in which the yield and recovery are highest.

Bioluminescence as a classroom tool for scientist volunteers.

There is a great need for practicing scientists to volunteer their time and expertise in the K-12th grade science classroom. We have found that bioluminescence is a fun and exciting way to teach basic science concepts and is an excellent tool for the volunteering scientist. We have had very positive reactions from both teachers and students. The excitement of the students when they first see bioluminescence is contagious. Bioluminescent dinoflagellates are one of the easiest ways to introduce students to this fascinating topic. Many activities and experiments can be done using the bioluminescent dinoflagellates and many students and teachers could benefit from your knowledge and expertise. See you in the classroom.

Specific immobilization of in vivo biotinylated bacterial luciferase and FMN:NAD(P)H oxidoreductase.

Bacterial bioluminescence, catalyzed by FMN:NAD(P)H oxidoreductase and luciferase, has been used as an analytical tool for quantitating the substrates of NAD(P)H-dependent enzymes. The development of inexpensive and sensitive biosensors based on bacterial bioluminescence would benefit from a method to immobilize the oxidoreductase and luciferase with high specific activity. Toward this end, oxidoreductase and luciferase were fused with a segment of biotin carboxy carrier protein and produced in Escherichia coli. The in vivo biotinylated luciferase and oxidoreductase were immobilized on avidin-conjugated agarose beads with little loss of activity. Coimmobilized enzymes had eight times higher bioluminescence activity than the free enzymes at low enzyme concentration and high NADH concentration. In addition, the immobilized enzymes were more stable than the free enzymes. This immobilization method is also useful to control enzyme orientation, which could increase the efficiency of sequentially operating enzymes like the oxidoreductase-luciferase system.

Thin-layer ion-exchange chromatography of proteins.

Thin-layer chromatography (TLC) is one of the simplest and most convenient techniques to separate small molecules. Of a variety of TLC separation modes, only size-exclusion was successfully used to separate proteins. In this paper, adsorption-TLC was used to separate proteins. The net charges were calculated for four model proteins, albumin, transferrin, lactoferrin and lysozyme, under different pH values. The suitable pH values for separation were determined according to the results from such calculations. Then, the adsorption isotherms of the four proteins were measured to deduce the ionic strength for appropriate elution conditions. Optimal conditions, 0.01 M bicine and pH 8.50, and a three-step elution process (1st step 0.01 M NaCl, 2nd 0.025 M NaCl, and 3rd 0.10 M NaCl), were obtained. Finally, the four model proteins were successfully separated under these elution condition.

Specific immobilization of firefly luciferase through a biotin carboxyl carrier protein domain.

Firefly luciferase (Photinus pyralis) was fused with a histidine tag and a biotin carboxyl carrier protein (BCCP) domain at its amino terminus. Highly purified recombinant luciferase was obtained by a one-step purification protocol, utilizing immobilized metal affinity chromatography. The novel BCCP-luciferase had properties, stability, and activity similar to those of native luciferase. The biotin molecule on the BCCP domain allowed specific immobilization of BCCP-luciferase on avidin-coated surfaces via the biotin-avidin interaction.

Minimizing interferences in the quantitative multielement analysis of trace elements in biological fluids by inductively coupled plasma mass spectrometry.

The determination of trace and ultratrace elements in biological fluids, including urine and serum, by inductively coupled plasma mass spectrometry (ICP-MS) is discussed. Nonspectral interferences and their corrections by external calibration and calibrator addition are discussed in detail. External calibration with internal calibration and dilution is mostly sufficient to correct for encountered biological matrix effects. For some elements, such as Cs and Zn, the use of calibrator addition provides more accurate results. The importance of spectral interferences and their elimination by isotope selection was also studied. Two examples, Cu and Zn, demonstrate the prime importance of selecting an isotope with minimal polyatomic interferences for analysis. By using 65Cu and 68Zn, accurate results for urine and serum can be obtained without excessive pretreatment of samples. Two reference materials, Bio-Rad Lyphochek urine and Kaulson Contox sera, were analyzed. Accuracy was evaluated by comparison with target values, and precision was estimated by the CV within 95% confidence.

Photoaffinity labeling of antibodies for applications in homogeneous fluoroimmunoassays.

A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and dansyl, were evaluated for this purpose. Also, this technology is ideal for fluorescence energy-transfer immunoassays that require labeling of the antibody with either a donor or acceptor fluorophore. In such cases, a fluorescent dye can be specifically attached near the antigen-combining site, where it can participate in high-efficiency energy transfer with its complementary fluorophore attached to the antigen.

Protein adsorption on low temperature isotropic carbon: V. How is it related to its blood compatibility?

Based on our research on blood protein interactions with low temperature isotropic carbon (LTIC) and data from the literature, we propose that the carbon surface has strong interactions with adsorbed proteins. In this paper we focus on how a relatively blood-compatible material interacts with plasma proteins. We present our results on the structure and properties of the LTIC surface utilizing SEM, STM, XPS, and contact angle measurements. We briefly review protein adsorption on LTIC using DSC, impedance, radioisotopes, and two-dimensional gel electrophoresis. LTIC is characterized by a microporous, oxidized, hydrophobic, and domain mosaic structure. Surface polishing smoothens the roughness and removes the porosity, while largely destroying the ordered atomic texture, making the surface more random and more amorphous. The LTIC surface denatures all adsorbed proteins studied. The rate of protein adsorption is high and the surface concentration is large. The LTIC surface adsorbs all proteins without preference. The surface also tenaciously retains proteins such that they cannot be displaced by buffer or exchanged by proteins in solution. We conclude that LTIC accomplishes its blood compatibility through a passivating film of strongly adsorbed bland proteins, which do not interact with platelets nor participate in blood coagulation. We also suggest mechanisms for the production of such a film by the LTIC surface.

Protein adsorption on low-temperature isotropic carbon: I. Protein conformational change probed by differential scanning calorimetry.

This is the first of a set of articles on protein adsorption on low-temperature isotropic carbon (LTIC), a reputed blood compatible material. Surface-induced conformational changes of albumin, fibrinogen, and some small proteins were measured by differential scanning calorimetry (DSC) on LTIC powders and colloidal silica. The LTIC surface significantly alters the DSC response (denaturation?) in proteins studied in different buffer solutions. We use the term "denaturation" to refer to altered protein behavior in the adsorbed state. Hydrophobic interactions between LTIC and the proteins are thought to be the major driving force. The presence of air at the water-carbon interface seems to prevent the surface denaturation of fibrinogen. The silica surface greatly denatures albumin but only slightly denatures fibrinogen. Because LTIC is considered to be a nonthrombogenic material, but silica is considered to be a thrombogenic one, whether a surface denatures adsorbed proteins cannot be the sole criterion for its blood compatibility. The latter largely depends on what protein the surface denatures, and in what sequences.

Protein adsorption on low temperature isotropic carbon. III. Isotherms, competitivity, desorption and exchange of human albumin and fibrinogen.

In this paper we consider the adsorption of albumin and fibrinogen on low temperature isotropic carbon (LTIC). A subsequent paper considers the adsorption of other plasma proteins [Feng L, Andrade JD, Colloids and Surfaces (in press)]. Carbon fragments and silica plates were used as adsorbents. Adsorption was carried out by incubating the adsorbents in solutions of 125I-labelled and unlabelled proteins (single component system), or with buffer-diluted human plasma (multicomponent system). Adsorbed proteins then underwent displacement by buffer, by single protein solutions or by dilute plasma. Results show that the LTIC substrate adsorbs a large amount of proteins before saturation, which may be due to multilayer adsorption. LTIC also irreversibly holds adsorbed proteins against the exchange agents used; little adsorbed proteins can be displaced, even after a very short adsorption time. There is no preferential adsorption for either albumin or fibrinogen on LTIC from their binary solutions, suggesting that both proteins have high affinities for the surface. Such strong interactions between LTIC and proteins are not attributed to electrostatic interactions. On the other hand, protein adsorption on the silica surface is selective and reversible, with a much higher affinity for fibrinogen than albumin and an even higher affinity for some other plasma proteins. The paper also discusses the effect of sequential protein addition to a solution on the surface concentration and suppression of adsorption of both proteins in the presence of other plasma proteins. A very important conclusion is that the LTIC surface is very active towards proteins adsorption.

Surface atomic and domain structures of biomedical carbons observed by scanning tunneling microscopy (STM).

STM has been used to study the surface domain and atomic structures of three biomedical carbons: glassy carbon (GC), low-temperature isotropic carbon (LTI), and ultra-low-temperature isotropic carbon (ULTI). The images show atomic lattices on both GC and LTI, but not on ULTI. The lattices contain many defects; lattices in GC are more ordered than those in LTI. The images also show patchlike carbon crystallites with sizes of 3-15 nm for GC, 2-8 nm for LTI, and 1-3 nm for ULTI. The crystallites from surface domains that may differ in surface properties due to different orientations of the crystallites. Mechanical polishing makes the LTI surface more amorphous and more homogeneous. Based on the STM observations, we evaluate several hypotheses on the blood compatibility of biomedical carbons.

Manipulation of Proteins on Mica by Atomic Force Microscopy.

The atomic force microscope was used to image adsorption of a monoclonal IgM on mica in real time. Under the smallest possible force we could achieve (<4 nN), the cantilever tip behaved as a molecular broom and was observed to orient protein aggregates in strands oriented perpendicularly to the facet of the cantilever tip. Rotating the scan direction preserved the orientational relationship, as seen by the formation of rotated strands. When the applied force was increased, the distance between the strands increased, indicating the amount of protein that can be swept depends on the applied force. The effect of scanning increased the apparent surface coverage of IgM. Manipulation of a deposited fibrinogen layer with a 4-nN repulsive force was observed only after tens of minutes, but not to the extent that strands formed, indicating a greater adhesion between the fibrinogen and mica than between IgM and mica. With an applied repulsive force of 30 nN, fibrinogen strands formed and the protein was manipulated to produce the block letter U. At a much higher repulsive force, the entire scanning area was swept clean.

Needs, problems, and opportunities in biomaterials and biocompatibility.

There are four topics related to biomaterials and biocompatibility which I feel are key problems, are often unrecognized, and are therefore rich opportunities for work in the near future: (i) the covalent instability of proteins, (ii) the concept of statistical specificity and statistical heterogeneity, (iii) the issue of solid surface dynamics and surface relaxation, and (iv) the growing concern with the costs of health care and of medical research. Each is briefly discussed in this paper.

Interaction of plasma proteins with heparinized gel particles studied by high-resolution two-dimensional gel electrophoresis.

In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.

Vroman effects, techniques, and philosophies.

Leo Vroman's work on blood-materials interaction over the years has motivated and influenced much of our work in this field. Here we show how most of our studies on proteins at interfaces can be traced to Vroman's ideas presented in Blood over 25 years ago. Specifically, we briefly discuss simple proteins at simple interfaces, complex interfaces, complex proteins at interfaces, multi-parameter phenomena, and scientific communication and education.

Adsorption of firefly luciferase at interfaces studied by total internal reflection fluorescence spectroscopy.

The adsorption of luciferase onto silica surfaces was studied by total internal reflection fluorescence (TIRF) spectroscopy. Two model surfaces were used: hydrophilic and hydrophobic silica. Luciferase adsorbed differently on these two surfaces. Initial kinetics of luciferase adsorption onto the hydrophilic surface showed that luciferase adsorbs over an adsorption energy barrier of ≈3 kT The quantum yield of luciferase fluorescence decreased at the hydrophilic silica surface, which indicated that the protein conformation was altered during adsorption. Luciferase adsorption onto the hydrophobic silica surface proceeded with a small adsorption energy barrier and the fluorescence efficiency of adsorbed protein remained unchanged after adsorption. The affinity of luciferase for luciferin was measured using quenching of luciferase fluorescence with luciferin. The binding constant of the adsorbed luciferase-luciferin complex at the hydrophilic silica surface was two orders of magnitude smaller than the respective binding constant in the solution. Adsorbed luciferase showed an absence of ATP-dependent visible luminescence, indicating that the adsorbed enzyme was not active at either of the two silica surfaces.