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Biomedical subjects

M A Marini

Publications and source records attributed to M A Marini.

At least 37 records · Page 2Linked to original sources
PubMed1990

High-performance liquid chromatographic evaluation of pyridoxyl 5'-phosphate hemoglobin derivatives produced by different reduction procedures.

Pyridoxylated hemoglobin derivatives have been studied by many investigators. In this study hemoglobin A0 rather than stroma-free hemoglobin was used as a starting material in order to reduce the number of proteins to A0 and A1c. Derivatives were characterized using a Synchropak Q300 strong anion-exchange column, a PolyCAT A weak cation-exchange column and a VYDAC reversed-phase high-performance liquid chromatographic column. Resulting peak profiles of these two ion-exchange separations demonstrated enhanced resolution and showed the presence of pyridoxylated hemoglobin products not previously described. We compared products from the reduction of these Schiff base derivatives using either sodium borohydride or sodium cyanoborohydride reduction procedures. The sodium cyanoborohydride reduction method produced a lower percentage of products with multiple-site pyridoxylation modifications than the sodium borohydride reduction process.

Blood Substitutes↗
PubMed1990

Coronary vasoconstrictor activity of purified and modified human hemoglobin.

Unpurified stroma-free hemoglobin (SFH) from water-lysed human red blood cells, hemoglobin Ao (HbAo), and hemoglobin cross-linked between alpha chains with 3,5-bis-dibromosalicyl-fumarate (HbXLDBBF) were infused into isolated perfused rabbit hearts. Vasoactivity and myocardial performance were determined using an isovolumic Langendorff preparation. With constant coronary flow, infusion of SFH (55 mg/dl) resulted in a 56% increase in aortic pressure as opposed to 29% and 11% increases with HbAo and HbXLDBBF, respectively. Rates of aortic pressure increase were over 6 times greater with SFH than with either HbAo or HbXLDBBF and exhibited concentration dependence only with SFH. Myocardial function remained normal. With constant coronary pressure, coronary flow decreased by 36% with SFH accompanied by a 23% decline in left ventricular developed pressure indicative of ischemia. With HbAo or HbXLDBBF, coronary flow decreased by only 1/3 that with SFH, while developed pressure declined by 4% and 2% with HbAo and HbXLDBBF, respectively. These data suggest that purification of HbAo to a single component can eliminate the dominant of at least two distinct vasoactive factors normally found in SFH. Furthermore, the physiological significance of the vascular response to HbAo is minimal. Preparations of HbXLDBBF accomplish this same result.

Animals↗
PubMed1990

Evidence for cooperative hydrogen ligand binding to sperm whale metmyoglobin.

Potentiometric titrations of sperm whale metmyoglobin from pH 10 to 3 shows the well-known exposure of groups between pH 4.5 and 4. However the reverse titration, at low protein concentration, results in the regeneration of its ionic property in the form of a reversible hysteresis, which was obtained by titrating the same solution twice. The Soret intensity band indicates reversibility for about 94%. Apomyoglobin shows an acid-base reversible titration from pH 9.5 to 3. Since the analysis by electrostatic interaction models do not adequately describe the overall ionic equilibria of metmyoglobin, a cooperative model was introduced. The model exactly reproduces the titration curve, indicating that 6 groups ionize with a cooperative coefficient of 9. The cooperative model explains the anomalous behaviour toward hydrogen ions and the incomplete spectral reversibility of sperm whale metmyoglobin as a molecular mechanism with physiological significance.

Animals↗
PubMed1989

Equilibrium oxygen binding to human hemoglobin cross-linked between the alpha chains by bis(3,5-dibromosalicyl) fumarate.

Oxygen equilibrium curves of human hemoglobin Ao (HbAo) and human hemoglobin cross-linked between the alpha chains (alpha alpha Hb) by bis(3,5-dibromosalicyl) fumarate were measured as a function of pH and chloride or organic phosphate concentration. Compared to HbAo, the oxygen affinity of alpha alpha Hb was lower, cooperativity was maintained, although slightly reduced, and all heterotropic effects were diminished. The major effect of alpha alpha-cross-linking appears to be a reduction of the oxygen affinity of R-state hemoglobin under all conditions. However, while the oxygen affinity of T-state alpha alpha Hb was slightly reduced at physiologic chloride concentration and in the absence of organic phosphates, KT was the same for both hemoglobins in the presence of 2,3-diphosphoglycerate (or high salt) and higher for alpha alpha Hb in the presence of inositol hexaphosphate. The reduced O2 affinity arises from smaller binding constants for both T- and R-state alpha alpha Hb rather than through stabilization of the low affinity conformation. All four Adair constants could be determined for alpha alpha Hb under most conditions, but a3 could not be resolved for HbAo without constraining a4, suggesting that the cross-link stabilizes triply ligated intermediates of hemoglobin.

2,3-Diphosphoglycerate↗
PubMed1989

Beta-endorphin receptors on cultured and freshly isolated lymphocytes from normal subjects.

[125I]-[D-Ala2]-beta EP*) binding to Epstein-Barr Virus (EBV) transformed B lymphocytes or to freshly isolated lymphocytes was characterized. The binding was time-, temperature- and pH-dependent; furthermore, it was reversible and cell concentration dependent. Maximum binding appeared at 15C in Tris buffer (pH 7.6) containing both BSA 0.5% and Bacitracin 0.1 mg/ml. beta EP inhibited BEP* binding to transformed lymphocytes and to freshly isolated lymphocytes by 50% at approximately 4 x 10(-8) M and 8 x 10(-9) M, respectively. Peptides representing amino-acid sequences 1-5, 1-16 and 1-17 of beta EP did not inhibit beta EP binding, neither did the opiates compounds Naloxone, Morphine, Bremazocine and Ethylketocyclazocine. On the contrary, beta EPd (6-31) inhibited beta EP* binding as effectively as beta EP, thus indicating that beta EP* binding to lymphocytes does not involve the N-terminal region of beta EP. 2 x 10(7)/ml freshly isolated lymphocytes bound 1.53 +/- 75%, whereas 2 x 10(6)/ml transformed lymphocytes bound 1.64 +/- 0.75% (mean +/- SD) beta EP* at tracer concentration. EBV transformed lymphocytes and freshly isolated lymphocytes may be useful to study beta EP receptors in humans.

Adrenocorticotropic Hormone↗
PubMed1989

A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao.

Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 +/- 2 torr and a Hill coefficient of 2.3 +/- 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen-ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5-phosphate (PLP) shows three major bands for the cyanoborohydride-reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed-bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 +/- 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse-phase chromatography indicates that the addition of PLP to HbAo is directed solely to the beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.

Hemoglobin A↗
PubMed1989

Inhibition of basal insulin secretion induces insulin resistance in normal man: evidence for a tonic effect of insulin on carbohydrate metabolism.

Insulin resistance has been demonstrated both in insulin deficiency and insulin excess in man and in animals. This study was carried out in normal man to evaluate the role of insulinopenia in the pathogenesis of insulin resistance. Insulin suppression was obtained by 4 h somatostatin (SRIF) infusion. Insulin receptors on circulating monocytes were evaluated before and after SRIF infusion; an insulin tolerance test (ITT) was performed after SRIF, saline or SRIF and replacing basal insulin secretion. Insulin binding to circulating monocytes did not change after 4 h insulinopenia (2.19 +/- 0.30 vs. 2.35 +/- 0.80%), while insulin sensitivity appeared decreased after SRIF (KITT = 0.97 +/- 0.13) as compared with saline (KITT = 3.30 +/- 0.42), and this effect was prevented by insulin (KITT = 2.46 +/- 0.38). A relationship was detected between KITT and plasma insulin concentration before ITT (r = 0.85, p less than 0.01), suggesting that insulin deficiency is the main cause of the phenomenon observed. The present data suggest that basal insulin concentration plays an essential role in the control of insulin sensitivity. If insulin binding on monocytes mimics the behavior of major insulin target tissues, it is possible that the impaired insulin action after 4 h of insulin deficiency is related to a post binding effect.

Adult↗
PubMed1988

Preparation of human hemoglobin Ao for possible use as a blood substitute.

A procedure is presented for the preparation of a purified fraction of adult human hemoglobin (HbAo) from one unit of outdated blood. The entire process requires less than 16 h and gives a sterile, endotoxin-free solution of HbAo (approximately 30 g) in a yield of 50%. The solutions are isoionic with a conductivity of less than 15 mu mhos and less than 2 mmol total phosphorus per mol heme. The methemoglobin content is less than 1% and on storage at 4 degrees C rises less than 1% per month.

Adult↗
PubMed1988

A convenient spectroscopic method for the estimation of hemoglobin concentrations in cell-free solutions.

Using a millimolar absorptivity of 7.12 +/- 0.09 at 523 nm, it is possible to estimate the total concentration of hemoglobin in solutions containing oxyhemoglobin, deoxyhemoglobin and methemoglobin in any combination. This estimate is independent of the pH in the range 6.0-10.0 and will provide concentrations comparable to that obtained by the conventional and more precise use of cyanomethemoglobin. This methodology should be of value for the determination of extracellular hemoglobin in vivo, as a means for determining the vascular half-life of stroma-free hemoglobin based blood substitutes.

Cell-Free System↗
PubMed1988

[Preparation of 125I-labelled monoclonal antibodies of the insulin receptor].

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.

Antibodies, Monoclonal↗
PubMed1986

Calorimetric studies of oxyhemoglobin ligand dissociation. III. Evidence for a hemoglobin-catalase-superoxide dismutase integrated system in the oxygen depletion by dithionite.

Calorimetric studies of the effect of superoxide dismutase and/or catalase on the reduction of dioxygen into water by dithionite in oxyhemoglobin have been carried out and the results compared with those in red cell hemolysates. In the absence of the enzymes the stoichiometry (moles dithionite/mole dioxygen) is less than the value of 2:1 which was found previously in red cell hemolysates [Forlani et al., J. Inorg. Biochem. 20, 147-155 (1984)]. In the presence of either superoxide dismutase or catalase alone the stoichiometry increases but is still less than 2:1. In the presence of both enzymes the stoichiometry and the shape of the thermogram is that previously observed for hemolysates, suggesting the presence of a hemoglobin-catalase-superoxide dismutase integrated system. The absence of a calorimetric signal for hydrogen peroxide in the reduction of oxyhemoglobin in the presence of superoxide dismutase suggests a wider biological role of superoxide dismutase than previously thought.

Catalase↗
PubMed1986

The determination of binding constants with a differential thermal and potentiometric titration apparatus. II. EDTA, EGTA and calcium.

A differential pH and thermal titrimeter has been used to determine the ionization constants of EDTA and EGTA as well as their calcium complexes. The intrinsic heat of binding is a constant for the pH range 2-11.5 for both substances and is found to be -5.4 kcal mol for EDTA and -7.9 kcal mol-1 for EGTA. The binding constants evaluated by fitting to the potentiometric curves and expressed as the log are 10.25 and 11.0, respectively. These values compare reasonably well to those reported in the literature. We have proposed that the binding of calcium occurs even at acid pH based on the pH drop observed at pH 2 when calcium is added, the reversibility of the thermal and the potentiometric curves, the lack of hysteresis upon rapid titration, the constancy of the intrinsic heat of binding and fitting of the entire potentiometric curves using the appropriate binding constants.

Calcium↗
PubMed1985

Calorimetric studies of oxyhemoglobin dissociation. II. Erythrocytic oxygen depletion by sodium dithionite.

Dithionite causes the depletion of dioxygen from suspensions of erythrocytes by reduction of the external dioxygen and not by diffusion into the cell. The molar enthalpy for the reduction shows a small difference with respect to the values found for free hemoglobin; and the normal stoichiometry of 2 moles dithionite/mole dioxygen found there is not observed with erythrocytes. At low hematocrit, the stoichiometry is 2.6:1 and decreases to 1.5:1 at high hematocrit. The change is not due to differences in the hemoglobin saturation or to an inability of dithionite to reduce all dioxygen present at the higher hematocrit. Neither catalase nor peroxidase added to the extracellular volume significantly alters the stoichiometry or the enthalpy of dioxygen reduction by dithionite. Addition of superoxide dismutase, however, restores the normal stoichiometry at high hematocrit and further increases the stoichiometry at low hematocrit. The calorimetrical signal of hydrogen peroxide, clearly seen with free dioxygen, is not present with erythrocytes. In all these cases the total heat evolved is the same.

Animals↗
PubMed1985

Application of the finite element simulation method to the adiabatic and potentiometric corrections of calorimetric titration data.

A general numerical analysis procedure is described which has been applied to an automated differential pH-thermal titration apparatus operated isoperibolically to obtain thermal corrections for heat loss. It is based on the Direct Byte (D-B) finite element computer simulation technique (FEST) applied to the heat conduction behavior of the instrument with time. Thermal constants of the numerical model are determined, and the results of the correction for titration data obtained from acid-base runs show that a constant upper baseline is achieved using this technique for both fast and slow reactions to an accuracy of 2%. The method is equally valid for endothermic and exothermic reactions.

Calorimetry↗
PubMed1985

Use of the twin-cell differential titration calorimeter for binding studies. I. EDTA and its calcium complex.

The use of a twin-cell differential titration calorimeter is described which utilizes small volumes (1-3 ml) of modest concentrations of materials (0.001-0.01 M) and yields data of good precision. Operation is controlled by a microprocessor which regulates and controls the addition of reagents and collects and displays the data as time, temperature in volts, and the pH. Corrections for the titration of water are applied to the potentiometric data, and the thermal data are corrected for the initial temperature-time baseline, the changes in heat capacity, and the heat loss (or gain) to the external environment. Finally, the thermal signal is corrected for the heat derived from the formation of water due to the free hydrogen or hydroxyl ions present. The corrected data as pH, groups titrated and delta HT (kcal/mol) can then be used to obtain the parameters pK' and delta Hi involved with the equilibria by curve-fitting the observed data. The system has been applied to the ionization of EDTA and its calcium complex. The ionization constants, the heats of ionization, the stoichiometry of binding and the heat of binding have been determined and demonstrated to be in agreement with published values.

Calcium↗
PubMed1985

Calorimetric and potentiometric studies on the binding of calcium by phytic acid.

Potentiometric as well as thermal titrations of phytic acid and its calcium complexes have been conducted using both the batch and titration microcalorimeters. For phytic acid, the experimental values by either method are in excellent agreement. For the calcium complexes, the total number of groups and the total heat evolved are in agreement but the placement of the curves is different due primarily to the differences in calcium concentration. Binding of calcium by phytic acid is endothermic for the pH range 2.0-12.0 while the heat of dilution of 1 M CaCl2 is exothermic. The binding at pH greater than 11 gives a value of 22.5 +/- 0.9 kcal mol-1. The same enthalpy of binding (22.3 +/- 0.6 kcal mol-1) could be calculated for the entire pH range studied which requires a knowledge of the observed heat of binding, the thermal titration curves of the acid and its calcium complex, and the change in the hydrogen ion environment. Inspection of the thermal binding curves at pH greater than 11 indicates that a number of step-binding constants are involved and that 5.2 mol calcium are bound/mole phytic acid. This value has been confirmed by atomic absorption spectroscopy. Both the thermal and the potentiometric curves are reversible either by the instantaneous injection of acid or base or by continuous titration. Values for the ionization constants (as pK') and the enthalpy of ionization (as delta Hi) have been estimated by computer assisted curve fitting.

Calcium↗