Metal complexes as allosteric effectors of human hemoglobin: an NMR study of the interaction of the gadolinium(III) bis(m-boroxyphenylamide)diethylenetriaminepentaacetic acid complex with human oxygenated and deoxygenated hemoglobin.

The boronic functionalities on the outer surface of the Gd(III) bis(m-boroxyphenylamide)DTPA complex (Gd(III)L) enable it to bind to fructosamine residues of oxygenated glycated human adult hemoglobin. The formation of the macromolecular adduct can be assessed by NMR spectroscopy via observation of the enhancement of the solvent water proton relaxation rate. Unexpectedly, a strong binding interaction was also observed for the oxygenated unglycated human adult hemoglobin, eventually displaying a much higher relaxation enhancement. From relaxation rate measurements it was found that two Gd(III)L complexes interact with one hemoglobin tetramer (KD = 1.0 x 10(-5) M and 4.6 x 10(-4) M, respectively), whereas no interaction has been observed with monomeric hemoproteins. A markedly higher affinity of the Gd(III)L complex has been observed for oxygenated and aquo-met human adult hemoglobin derivatives with respect to the corresponding deoxy derivative. Upon binding, a net change in the quaternary structure of hemoglobin has been assessed by monitoring the changes in the high-resolution 1H-NMR spectrum of the protein as well as in the Soret absorption band. On the basis of these observations and the 11B NMR results obtained with the diamagnetic La(III)L complex, we suggest that the interaction between the lanthanide complex and deoxygenated, oxygenated, and aquo-met derivatives of human adult hemoglobin takes place at the 2, 3-diphosphoglycerate (DPG) binding site, through the formation of N-->B coordinative bonds at His143beta and His2beta residues of different beta-chains. The stronger binding to the oxygenated form is then responsible for a shift of the allosteric equilibrium toward the high-affinity R-state. Accordingly, Gd(III)L affinity for oxygenated human fetal hemoglobin (lacking His143beta) is significantly lower than that observed for the unglycated human adult tetramer.

NMR relaxometric investigation on human methemoglobin and fluoromethemoglobin. An improved quantitative in vitro assay of human methemoglobin.

Longitudinal water proton relaxation rates of methemoglobin solutions show a strong dependence on temperature and pH. The increase of the relaxation rates with temperature is associated with shortened exchange lifetime of the coordinated water molecule. An accurate measurement of the relaxation rate of methemoglobin solutions thus requires careful control of the experimental temperature. This observation prompted the authors to look for an improved version of the relaxometric in vitro determination of methemoglobin. The method is based on transforming methemoglobin into the corresponding fluoromethemoglobin derivative, which shows both a higher relaxivity and a negligible dependence on temperature. The proposed method has been found to be in good agreement with data from spectrophotometric assays.

Association of pseudothrombocytopenia and pseudoleukopenia: evidence for different pathogenic mechanisms.

A case of spurious automated leukocyte and platelet counts due to EDTA-dependent platelet-neutrophil agglutination is described. Whole blood anticoagulated with K3EDTA, sodium citrate and lithium heparin was analysed at short time intervals up to 6 h from sampling at 37 degrees C and at room temperature (RT = 27 degrees C). A phenomenon of platelet clumping occurred at both temperatures with all the anticoagulants (pseudothrombocytopenia), while platelet-granulocyte agglutination was present only with EDTA at RT. Aggregates consisting of up to 80 neutrophils were seen on the blood smear. The contemporary presence of platelet clumping caused a reduction of WBC count of only 25% the initial, while leukocyte differential was markedly altered (pseudolymphocytosis). Further experiments were performed mixing plasma and serum of the patient with packed cells (PC) from a normal donor in the presence of different anticoagulants and at various dilutions and temperatures. Platelet-neutrophil agglutination occurred only in the presence of EDTA at temperatures lower than 37 degrees C, and was abolished by plasma dilutions from 1:8 onwards. Similarly, it was inhibited by incubation with dithiothreitol (DTT), in contrast with platelet clumping. The latter phenomenon was triggered by an EDTA concentration lower than that necessary to cause platelet-neutrophil agglutination (0.5 mg ml-1 vs. 0.77 mg ml-1). Obtained results suggest the causal association of 2 different phenomena, both transferable to normal cells by means of patient plasma and serum. In the article the pathogenetic implications of the case are discussed.

Quantitative determination of methemoglobin by measuring the solvent-water proton-nuclear magnetic resonance relaxation rate.

We report a new method for the quantitative determination of human methemoglobin (metHb) based on the measurement of the solvent-water proton-nuclear magnetic resonance (NMR) relaxation rate R1 [normalized to 1 mmol/L hemoglobin (Hb) concentration]. MetHb (%) is estimated from the linear dependence of R1 on the metHb concentration, taking into account the simple relationship [MetHb] = [(R1 - R1HbO2)/(R1metHb - R1HbO2)].100, where R1HbO2 and R1metHb are values for the solvent-water relaxation rate of standard 1.0 mmol/L solutions of the oxygenated derivative of human hemoglobin (HbO2) and of metHb, respectively. The minimum metHb that may be determined from the analysis of the experimental data is 0.5 +/- 0.4%.