Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis.

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.

Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin.

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. An affinity capillary electrophoresis method has been developed to evidence the complex formation and to determine the binding properties between an anticoagulant polysaccharide of marine origin, fucoidan, and a potential target protein, antithrombin. This method is a variant of zonal electrophoresis in the mobility shift format. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant for the polysaccharide/antithrombin complex. The results indicate that fucoidan binds to antithrombin in a 1:1 stoichiometry and with an affinity depending on the molecular weight of the polysaccharide. For heparin, the binding constant obtained similarly is in accordance with the literature. This is the first report showing the implementation of a capillary electrophoresis method contributing to the mechanistic understanding of the biological activities of fucoidan and providing evidence for the complex formation between fucoidan and the protein inhibitor of the coagulation antithrombin.

Regioselective desulfation of sulfated L-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus: application to the structural study of algal fucoidan (Ascophyllum nodosum).

The study of the structural bases of the biological properties of algal fucoidan (Ascophyllum nodosum) led us to look for enzymes able to modify this sulfated polysaccharide. In this context, we found a sulfoesterase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on fucoidan. This sulfoesterase activity was shown by capillary electrophoresis and 13C-1H NMR (500 MHz) analysis of the enzymatic hydrolysis of the fucoidan, of fucoidan oligosaccharides and of sulfated fucose isomers. We report the exhaustive list of all proton and carbon chemical shifts for each of the three isomers of sulfated-l-fucose (including of their alpha/beta anomers), i.e. the 2-O-, 3-O- and 4-O-sulfated fucose, which have been useful reference values for the assignments of NMR spectra of fucoidan oligosaccharides upon enzymatic desulfation. Our results demonstrated a high regioselectivity for this sulfoesterase, which hydrolyzes only the sulfate group at the 2-O position of the fucopyranoside. Therefore, this sulfoesterase is a helpful tool in the structure-activity study of the fucoidan, as the literature data suggest that the 2-O-sulfation level play a central role in the biological properties of the polysaccharide.

Quantitative analysis of mixtures of metal-carbonyl complexes by Fourier-transform infrared spectroscopy: application to the simultaneous double immunoassay of antiepileptic drugs by the nonisotopic carbonyl metalloimmunoassay method.

The feasibility of a double immunoassay of haptens by the nonisotopic carbonyl metalloimmunoassay (CMIA) method is demonstrated. Three different pairings of antiepileptic medications from the groups carbamazepine, diphenylhydantoin, and phenobarbital (for each of which a mono-CMIA is already available) were assayed by double CMIA. The assay method employs as tracers metal-carbonyl complexes that give very strong signals in the range of 1850-2200 cm-1 in the infrared spectrum, permitting quantitative analysis by Fourier-transform infrared spectroscopy. The fact that the signals are individually assignable and of comparable intensity permits quantitative analysis of mixtures of two tracers. The analysis may proceed in one of two ways: in the simpler case, there is no peak overlap with the two tracers and the quantitative analysis can be performed by simply measuring the absorbance of characteristic peaks of the two tracers. In the second case, in which there is partial or total overlap of peaks, a stepwise calculation provides rapid quantification of the two tracers. These findings allowed us to perform the double CMIA of two antiepileptics in which experimental conditions and time of analysis were strictly identical to those for mono-CMIA. We show here that there is a very good correlation between the results obtained in mono- and double-immunoassay by the CMIA method (correlation coefficient > 0.990).

Production of specific antibodies and development of a non-isotopic immunoassay for carbamazepine by the carbonyl metallo-immunoassay (CMIA) method.

As part of our ongoing work to extend the range of applications of the non-isotopic carbonyl metalloimmunoassay (CMIA), previously developed in our laboratory, we describe here the first CMIA study of carbamazepine. The CMIA method uses a metal carbonyl complex as a non-isotopic tracer, and in this case we chose to employ the dicobalt hexacarbonyl moiety (Co2(CO)6) attached to an alkyne. Two organometallic tracers, 3 and 7, were synthesized, differentiated by the nature and length of the spacer arm of the Co2(CO)6 moiety. Two different coupling methods were subsequently used to synthesize the immunogens 1 and 2, the first one used a carbodiimide, while the second, employed dimethyl adipimidate as coupling agent. Titer values of the antisera obtained by injection of these immunogens into rabbits, were determined by CMIA, using one of the organometallic complexes, 3 or 7, as tracer. Both antisera had higher titer values with the long-chain tracer, 7, than with the short-chain tracer, 3. However these titer values were very different: low for antiserum 1 and high for antiserum 2. The cross-reactivity of antiserum 2 with other antiepileptic drugs was negligible. For competition curves, there was good sensitivity with the antibody 2/3 pairing, while a broad assay range was obtained with antibody 2/7 pairing. These results demonstrate the viability of CMIA as an immunoassay method for carbamazepine, and open the way to development of a simultaneous multiassay by CMIA of the principal antiepileptic drugs.

Application of the non-radioisotopic carbonyl metalloimmunoassay (CMIA) to diphenylhydantoin.

As part of our ongoing work to develop the new non-isotopic assay method carbonyl metalloimmunoassay (CMIA), whose efficacy has already been proven in the laboratory for phenobarbital and cortisol, we here present the steps involved in establishing CMIA of 5,5 diphenylhydantoin (DPH), one of the most commonly used antiepileptic medications. First, anti-DPH antibodies were obtained by injection of the immunogen DPH-3-valerate-BSA into rabbits. The titer value and specificity of these antibodies were examined by RIA using [14C]-DPH as tracer, and an antibody batch selected for its high titer value and good specificity for metabolites of DPH and other antiepileptic drugs. Next the organometallic complex Cr(CO)3-DPH, chosen as the CMIA tracer, was synthesized and shown to conserve a high recognition value for anti-DPH antibodies (CR = 200%). Isopropyl ether was selected as the best organic solvent for use in separating the free and bound fractions of the tracer. Employing the Cr(CO)3-DPH complex as tracer and FT-IR spectroscopy as the detection method, we were able to obtain a titration curve by CMIA using an amount of tracer identical to that used in RIA. The titer value obtained in CMIA is approximately twice that obtained by RIA. These results demonstrate the feasibility of DPH assay by the CMIA method.

Interatrial conduction in patients undergoing AV stimulation: effects of increasing right atrial stimulation rate.

To evaluate the frequency of spontaneous or rate dependent interatrial blocks, the interatrial conduction time (IACT) was studied on 100 consecutive patients (mean age 78.3 +/- 7.8 years) during a transvenous dual chamber pacemaker implant. The spontaneous interatrial conduction time (SIACT) was measured from the intrinsic deflection (ID) of the unipolar right atrial signal to the ID of the left atrial signal recorded in a bipolar way by an esophageal lead. The paced interatrial conduction time (PIACT) was measured from the stimulus artifact to the left atrial ID, when the atrium was paced at a slightly higher rate than the spontaneous rate and during incremental atrial pacing. From these measurements, the maximum increase of PIACT (MIPIACT) was deduced. In this elderly population, the PIACT was similar (117 +/- 26.9 msec) to the data in the literature. However, there were large interindividual variations that were also found in SIACT. We found a close correlation between SIACT and PIACT (P < 0.0001). PIACT was on average 50 msec longer than SIACT. SIACT increased with age (P < 0.03). The MIPIACT was 15.3 +/- 15.2 msec. In the majority of patients, the MIPIACT was > 10 msec, and even reached 90 msec in one patient. MIPIACT was longer in patients with a PIACT exceeding 110 msec (P < 0.004). Based on IACT alone, the AV interval must be lengthened on average by 50 msec when changing from atrial tracking-ventricular pacing to atrial pacing-ventricular pacing, but large individual differences must be kept in mind. Elderly people should probably have a longer AV delay.

Transition metal carbonyl labeling of proteins. A novel approach to a solid-phase two-site immunoassay using Fourier transform infrared spectroscopy.

Labeling of bovine serum albumin (BSA) and anti-human thyroid stimulating hormone (hTSH) monoclonal antibodies (mAbs) was performed using (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt (NSCo2(CO)6). Conditions of coupling were different depending on the protein to be labeled, denaturation of the mAbs occuring with high percentages of organic solvent in the reaction mixture. The influence of reaction time and initial concentration of NSCo2(CO)6 was examined. They were both shown to affect the final coupling rate of the metal carbonyl probe. Preservation of the immunoreactivity toward 125I-hTSH was observed for five conjugates having different NSCo2(CO)6: mAb molar ratios when compared to unmodified and peroxidase-labeled mAbs. Finally, a preliminary study of the quantitative detection of the metal carbonyl mAbs on microtiter wells was achieved using Fourier transform infrared spectroscopy.

[Propafenone in ventricular extrasystole in elderly patients. Tolerance and efficacy]. / La propafénone dans l'extrasystolie ventriculaire du sujet âgé. Tolérance et efficacité.

The tolerance and efficacy of propafenone were studied in cases of chronic ventricular extrasystoles of the elderly (age greater than 70 years). Only patients presenting more than 1,000 extrasystoles per day and requiring a treatment, were included in this study. Patients presenting non-compensated cardiac insufficiency, hypotensive patients, patients with conduction disorders without pacemakers as well as patients presenting a severe renal or hepatic insufficiency, were excluded. After making sure that the rhythm disorder was chronic, a control Holter was performed, then, 600 mg of propafenone per day, in 3 doses were prescribed for 5 days. On the 5th day, a Holter was performed as well as a blood propafenone level. Some patients received then 900 mg per day with a new Holter and plasma propafenone titration on the 10th day. 11 patients were studied in this fashion. The tolerance to 600 mg was good in 10 out of 11 cases. The efficacy, appreciated by the decrease of the number of isolated ventricular extrasystoles (-69%), doublets (-95%), triplets (-98%) and salvos of ventricular tachycardias (-100%), was excellent in 6 cases, average in 2 cases and mediocre in 3 cases. Increase of the dosage to 900 mg does not improve significantly the anti-arrhythmic efficacy while the side effects seem more frequent. It is not possible to establish a relationship between plasma concentrations and efficacy. At a dosage of 600 mg per day, propafenone is therefore effective in the treatment of ventricular extrasystoles in elderly patients and its tolerance is good. Higher doses are not advisable as they seem much less well tolerated without any additional advantage.

[Surface recording of abnormal post-QRS micropotentials. Description and frequency in ventricular tachycardias after myocardial infarction]. / Enregistrement de surface des micropotentiels anormaux post-QRS. Description et fréquence dans les tachycardies ventriculaires après infarctus du myocarde.

In order to study abnormal post-QRS micropotentials, so called late potentials, and to determine their frequency in post myocardial infarction ventricular tachycardia (VT), high amplification electrocardiogrammes were recorded in 180 patients classified in 3 different groups: Group A comprising 36 patients who developed sustained VT after myocardial infarction; Group B comprising 124 patients with myocardial infarction uncomplicated by VT. This group was subdivided into subgroup B1 (retrospective study of 35 patients with chronic myocardial infarction, dating on average 10 months--range 7 days to 8 years) and subgroup B2 (prospective study of 89 patients investigated on the 7th and 60th days after infarction); Group C comprising 20 young, normal control subjects. Using computer assisted high amplification electrocardiography, all patients underwent at least 3 successive recordings of the following parameters: averaging 100 cycles; sampling: 1 kHz; band pass 20-300 Hz and 80-300 Hz; gain setting 10,000 and 25,000. Late potentials usually appears, after the end of the S wave, as high frequency oscillations with an amplitude (10 to 20 microV) significantly greater than that of the background noise. Our recordings also showed: the frequent presence, especially in intraventricular blocks, of fragmentation of the end of the R wave and of the S wave or terminal potentials; the presence of an abnormal giant low frequency high amplitude wave (40 to 80 microV) in 5 patients with a large left ventricular aneurysm.(ABSTRACT TRUNCATED AT 250 WORDS)

[Implantation of permanent cardiac pacing electrodes by the retropectoral transvenous approach. Results of a series of 152 cases]. / Mise en place des sondes de stimulation cardiaque permanente par les veines rétro-pectorales. Résultats d'une série de 152 cas.

When the cephalic vein is unsuitable for the introduction of pacing electrodes, the retropectoral veins near the external border of pectoralis major near its subclavian attachment, approached through the same incision, may provide a suitable alternative. It was not possible to catheterise the cephalic vein in 23,8% of 756 consecutive implantations of endocavitary pacing electrodes. The retropectoral veins were looked for in 172 cases and found and used in 159 cases (92,4%). This percentage of success increased to 97,6% in the latter 83 attempts. These veins are usually very distensible. No complications or accidents were recorded. The only disadvantage was the relatively long dissection time. The stability of the pacing electrodes with this approach was excellent as reoperation was only required in 3% of cases (2 displacements and 3 exit blocks or pericardial migrations). This approach is therefore practicable in the large majority of cases in which the cephalic vein cannot be used. The multiplicity of the retropectoral veins should allow the introduction of two electrodes if sequential atrioventricular pacing were to be chosen. In addition, this approach would be useful when an atrial pacing electrode is to be added to a preexisting ventricular pacing electrode and one hesitates to puncture the subclavian vein because of the risk of damaging the electrode already in place. When direct subclavian puncture is the technique of choice of the operator, the retropectoral veins may be used when the subclavian approach is contraindicated or impossible. In any case, denudation of the retropectoral veins leads to fewer incidents than when the latter approach is used.

[Aortic insufficiency and syphilitic coronary ostial disease. Pre- and postoperative angiographic aspects]. / Insuffisance aortique et coronarite ostiale syphilitique. Aspects angiographiques pré et postopératoires.

The authors report the case of a 50 year old patient with severe aortic incompetence (stage IV on angiography) and syphilitic left coronary ostial disease confirmed by complementary investigations. Management comprised aortic valve replacement and saphenous vein aorto-coronary bypass. The postoperative angiographic appearances showed normal valve function and a permeable aorto-left anterior descending bypass graft. The outcome was also satisfactory from the clinical point of view.

[Aneurysm of the interauricular septum by hernia of Vieussens' valve, and suspected coronary embolism]. / Anévrysme de la cloison inter-auriculaire par hernie de la valvule de Vieussens et suspicion d'embolie coronaire.

During the hemodynamic evaluation of a young patient with heart failure whose electrocardiogram showed sequels of anterior necrosis, an aneurysm of the fossa ovalis was seen at cineangiography. Selective coronary angiography showed an interruption of filling of the anterior interventricular artery just distal to the branching of the first septal artery: it raised therefore the possibility of the embolic origin of that myocardial infarction. In connection with this case study, angiographic aspect, pathogenesis and the embolic role of the aneurysm of the fossa ovalis were discussed.