Structural analysis and corrosion studies on an ISO 5832-9 biomedical alloy with TiO2 sol-gel layers.

The aim of this study was to demonstrate the relationship between the structural and corrosion properties of an ISO 5832-9 biomedical alloy modified with titanium dioxide (TiO2) layers. These layers were obtained via the sol-gel method by acid-catalyzed hydrolysis of titanium isopropoxide in isopropanol solution. To obtain TiO2 layers with different structural properties, the coated samples were annealed at temperatures of 200, 300, 400, 450, 500, 600 and 800 °C for 2 h. For all the prepared samples, accelerated corrosion measurements were performed in Tyrode's physiological solution using electrochemical methods. The most important corrosion parameters were determined: corrosion potential, polarization resistance, corrosion rate, breakdown and repassivation potentials. Corrosion damage was analyzed using scanning electron microscopy. Structural analysis was carried out for selected TiO2 coatings annealed at 200, 400, 600 and 800 °C. In addition, the morphology, chemical composition, crystallinity, thickness and density of the deposited TiO2 layers were determined using suitable electron and X-ray measurement methods. It was shown that the structure and character of interactions between substrate and deposited TiO2 layers depended on annealing temperature. All the obtained TiO2 coatings exhibit anticorrosion properties, but these properties are related to the crystalline structure and character of substrate-layer interaction. From the point of view of corrosion, the best TiO2 sol-gel coatings for stainless steel intended for biomedical applications seem to be those obtained at 400 °C.

[Angiotensin I-converting enzyme in cerebrospinal fluid and neurosarcoidosis]. / Enzyme de conversion de l'angiotensine I dans le liquide céphalorachidien et neurosarcoïdose.

Sarcoidosis is a disease of unknown aetiology. This granulomatous disease is essentially localized in lung and skin, but many other localizations are possible, such as in nervous system. Sometimes the neurological involvement is alone leading to a differential diagnosis from other neurological diseases. Angiotensin I-converting enzyme (ACE) is synthesized by sarcoidotic granulomas and diffuses in various biological fluids. The determination of ACE activity in cerebrospinal fluid (CSF) can help for the diagnosis of neurosarcoidosis, associated or not to its determination in serum. We developed a radiometric assay for the determination of ACE activity in CSF since the methods for serum cannot be used because ACE activity is low in CSF, as well as in pathological situations. At the analytical point of view this assay is sensitive, specific and reproducible. We established a normal range and yielded recommendations to give the results, particularly in function of the aspect of the CSF and the proteinorrachia. But increased level of ACE in CSF is not specific of neurosarcoidosis since elevations were also shown in meningitis. We can claim for the routine use of ACE assay in CSF for differential diagnosis by eliminating neurosarcoidosis, as well as for positive diagnosis of this disease, but in both cases with the confrontation to other parameters.

Anticancer drugs induce necrosis of human endothelial cells involving both oncosis and apoptosis.

The endothelium is the first physiological barrier between blood and tissues and can be injured by physical or chemical stress, particularly by the drugs used in cancer therapy. We found that four anticancer agents: etoposide, doxorubicin, bleomycin and paclitaxel induced apoptosis in human umbilical vein endothelial cells (HUVECs) (as judged by DNA fragmentation) with a time- and concentration-dependent decrease in bcl-2 protein but without the involvement of p53. As revealed by immunoblotting, bax protein was expressed in HUVECs treated with 1 mg/ml etoposide whereas bcl-2 protein disappeared. Oncosis occurred parallel to apoptosis with the release of lactate dehydrogenase into the supernatant, and, for doxorubicin and etoposide with the inversion of the distribution of angiotensin I-converting enzyme between supernatant and cells. Among the four tested anticancer drugs, only doxorubicin induced an oxidative stress, with significative malondialdehyde production. Thus, human endothelial cells in confluent cultures seem to be in an equilibrium of resistance to apoptosis related to bcl-2 expression; this equilibrium can be disrupted by a chemical stress, such as the antiproliferative drugs known as pro-apoptotic for tumour cells. For doxorubicin and bleomycin, this cellular toxicity can be related to their unwanted effects in human cancer therapy. Low doses of doxorubicin, paclitaxel or etoposide, however, could induce apoptosis of endothelial cells of new vessels surrounding the tumour, thus leading to specific vessel regression with minimal toxic effects for the endothelium of the other vessels. These findings provide evidence of relationships between endothelial toxicity of anticancer drugs and the key role of bcl-2 for resistance of endothelium cells toward apoptosis; moreover lack of p53 and bax in quiescent cells contributes to resistance of endothelial cells to DNA-damaging agents.

Evaluation of Stratus CS stat fluorimetric analyser for measurement of cardiac markers Troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin.

Myoglobin, CK-MB, and Troponin I (cTnI) are cardiac muscle necrosis markers that are useful for detecting acute myocardial infarction (AMI). The Stratus CS (Dade Behring, Inc.) is a discrete fluorimetric immunoassay analyser designed for the determination of the three cardiac markers from a single sample of whole blood or plasma. Overall analytical performances of the Stratus CS provided by Dade Behring were evaluated according to the French Society of Clinical Biology guidelines. Within-run imprecision (n = 20) for the three parameters at three levels gave values under 5%, whereas CVs for between-run imprecision (n = 20) were under 6%. The sensitivities were 0.03 microg/L for cTnI and 0.4 microg/L for CK-MB. Linearities extended from 0-50 microg/L for cTnI, 0-140 microg/L for CK-MB, and 1-900 microg/L for myoglobin. The results, particularly those obtained on whole-blood samples, correlated well with those obtained on Stratus II. We did not find any interference with haemolysis, icterus, or lipemia. The system was very easy to use, and fulfills the requirements for the analysis of the three cardiac markers in patients with acute chest pain in emergency situations.

Mixed-type inhibition of pulmonary angiotensin I-converting enzyme by captopril, enalaprilat and ramiprilat.

We have compared at the enzymological level pulmonary angiotensin I-converting enzymes (ACE) purified to electrophoretic homogeneity from four mammalians species: pig, rat, monkey and human. Using both substrates hippuryl-histidyl-leucine and furylacryloyl-phenylalanyl-glycyl-glycine in steady-state conditions, all the ACEs exhibited Michaelis kinetics with identical Michaelis constants, maximal velocities, optimal pH and optimal activating chloride-concentrations. The apparent inhibitory constant was higher for Captopril than for Enalaprilat and even more so for Ramiprilat irrespective of the origin of ACE and the substrate used. Although these inhibitors have been described as competitive inhibitors, Lineweaver-Burk plots were not in accordance with a simple competitive model; moreover, Dixon plots were rather characteristic of non-competitive inhibition. These data emphasize the hypothesis that ACE inhibitors act with mixed-type inhibition, which is consistent with their slow-tight binding to the ACE active center, also with binding of chloride on a critical lysine residue leading to a potential conformational change, and finally with the fact that ACE has two domains, each bearing one catalytic site. On the other hand, as identical kinetic parameters were obtained on the different ACE preparations, results from animal models should allow the extrapolation to humans, in particular for investigations on both renin-angiotensin and kallikrein-kinin systems, and on their inhibition.

Plasma concentration, kinetic constants, and gene polymorphism of angiotensin I-converting enzyme in centenarians.

We have determined serum activity and kinetic constants of angiotensin I-converting enzyme (ACE), parallel to an insertion/deletion (I/D) polymorphism in its gene, in French centenarians and controls 20-70 years of age because this enzyme could have an impact on cardiovascular risk, and thus on longevity. Both the ACE D allele and ACE D/D genotype were more frequent in centenarians in comparison with controls, without sex-related differences nor significant correlation with a cardiovascular pathology. In centenarians, I/D polymorphism was correlated with circulating ACE activity (D/D genotype, 89.0 +/- 36.8 U/L; I/D genotype, 63.5 +/- 26.0 U/L; and I/I genotype, 55.1 +/- 39.4 U/L). The Michaelis constants for two substrates were identical whatever the genotype and were not different between centenarians and controls, i.e., 0.30 +/- 0.03 mmol/L for furylacryloyl-phenylalanyl-glycyl-glycine and 1.35 +/- 0.05 mmol/L for hippuryl-histidyl-leucine; for the latter, the optimal pH and activating concentration of chloride did not depend on I/D polymorphism. The maximal velocities with both substrates reflected the distribution of serum ACE activity as a function of the genotypes, in centenarians and in controls. In conclusion, plasma ACE activity is subject to a similar genotypic influence in centenarians as in adults 20-70 years of age; however, ACE itself appears to be functionally similar for each genotype. Furthermore, the D allele as well as the higher serum ACE activities associated with the D/D genotype cannot discriminate individuals at high risk for cardiovascular diseases, major causes of mortality before the age of 100 years.

Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme.

We enzymatically deglycosylated pig lung angiotensin I-converting enzyme (ACE) to study the involvement of its glycanic chains in its physicochemical and catalytic properties. The effects of endoglycosidases F2 and H, and of N-glycanase were assessed by ACE mobility in SDS-PAGE. N-Glycanase only was completely effective with or without previous denaturation, leading to a shift in ACE M(r) from 172 to 135 kDa; endoglycosidase F2 produced the same shift but only without previous denaturation. Deglycosylated ACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine, and an identical Stokes radius as measured by size-exclusion high performance liquid chromatography. Neuraminidase had no effect on ACE Stokes radius but slightly decreased its kcat which could be related to variations in ionization of the active site. The isoelectric point of ACE, as, determined by isoelectric focusing, increased from 4.5-4.8 to 5.0-5.3 after either endoglycosidase F2 or neuraminidase digestion, but still with microheterogeneities which thus did not seem to be related to ACE glycans. Deglycosylated ACE did not bind onto agaroselectins in contrast to native ACE which bound strongly to concanavalin A showing interactions involving oligomannosidic or biantennary and sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin, an inhibitor of N-glycosylation, did not modify ACE secretion by endothelial cells. Thus, ACE glycans have no drastic effects on structural and biological properties of the protein, but they may have a functional role on intracellular targeting of both secreted and membrane-bound ACE isoforms, also for the protection of the soluble plasma form against hepatic lectins and the maintenance of its hydrosolubility.

Cytotoxicity of amiodarone in cultured human endothelial cells.

As damage to the pulmonary vascular endothelium may be responsible for the lung toxicity of amiodarone, we evaluated the cytolytic toxicity of the drug in cultures of endothelial cells. Cells were cultured from human umbilical cord veins. Amiodarone caused a vacuolization of the cells with liberation of both lactate dehydrogenase (LDH) and angiotensin-converting enzyme (ACE) in the culture medium. These effect were both concentration and time dependent, and were correlated between them. The first toxic effects were shown as soon as 2 hours after contact with the drug and at 0.1 mg/ml, a concentration that can be reached in plasma of amiodarone-treated patients. A decrease of ACE activity in the cells was delayed to 24 hours and only with the 10 mg/ml concentration. This event correlated with cell death and detachment from the extracellular matrix. LDH increases corresponded to its isoenzymes 3 and 4. These data support the hypothesis of a direct toxic effect of amiodarone on the endothelium and show the need for evaluating LDH, total activity and isoenzymic profile, and ACE determinations in the plasma of patients treated with amiodarone for ischemic heart disease or arrhythmia.

Physiochemical and immunological comparisons between angiotensin I-converting enzymes purified from different mammalian species.

Angiotensin I-converting enzyme (ACE) was purified from lungs of pig, rat, monkey and human for comparison of its physicochemical, enzymatic and immunological properties. The protocol involved three chromatographic steps after detergent extraction, i.e. DEAE-Sphérodex ion exchange, lisinopril-Sepharose affinity and Superose 12 HPLC, plus Mono-Q HPLC for monkey ACE. Purified ACE's presented numerous homologies: in particular, closely similar specific activities, catalytic efficiencies, Km's, optimal pH and chloride activations; the molecular weights were about 170 kDa by SDS-PAGE and 320 kDa by gel-filtration on Superose 12; the isoelectric points were about 4.5-4.7. Specific polyclonal antibodies recognized the antigen (porcine ACE) as well as rat, monkey and human ACEs. In contrast, three monoclonal antibodies (F02.4.1, F01.1.3 and F03) produced against porcine ACE showed some differences: they only reacted with pig enzyme and only one (F0.2.4.1) was anticatalytic. Moreover, the cross-reactivity judged on ELISA with porcine ACE characterized different epitopes specific for the porcine enzyme. In particular, the binding of F02.4.1 was not diminished by previous treatment with saturating concentrations of synthetic competitive ACE inhibitors. Thus, the extrapolation to human of data obtained on animal models should be possible at least for pharmacological and medical trials.

Plasma fibronectin and angiotensin-converting enzyme: markers of primary pulmonary injury in burn patients.

Plasma fibronectin (FBN) and angiotensin I-converting enzyme (ACE) were prospectively measured in 50 burn patients from the day of admission to day 28 after the trauma with the aim of finding biochemical markers of pulmonary injury by smoke inhalation. Patients were divided into three groups on the basis of fiberoptic bronchoscopy results (group I: healthy lungs; group II: burned lungs; group III: infected lungs). A decrease in FBN concentrations was observed in the three groups but was larger in group II than in the other groups, especially at day 2 (P < 0.05). A similar profile was observed for plasma ACE activity. Factors other than lung injury may influence plasma FBN and ACE levels, in particular the burned body surface area, an acute event such as septicemia, or outcome. However, repeated measurements of both markers could help in the assessment of lung injury in the follow-up of burn patients.

Angiotensin-converting enzyme: clinical applications and laboratory investigations on serum and other biological fluids.

Angiotensin I-converting enzyme (ACE) is a peptidyldipeptide hydrolase that is located mainly on the luminal surface of vascular endothelial cells but also in cells derived from the monocyte-macrophage system. Physiologically, ACE is a key enzyme in the renin-angiotensin system, converting angiotensin I into the potent vasopressor angiotensin II and also inactivating the vasodilator bradykinin. Increased serum ACE activity (SACE) has been reported in pathologies involving a stimulation of the monocytic cell line, primarily granulomatous diseases. Sarcoidosis is the most frequent and the better studied of these diseases; high SACE is not only a well-established marker for the diagnosis but is also a useful tool for following its course and evaluating the effect of therapy. SACE can also be increased in nonsarcoidotic pulmonary granulomatous diseases such as silicosis and asbestosis, in extrathoracic granulomatous pathologies such as Gauchers disease and leprosis, and, to a lesser extent, in nongranulomatous disorders such as hyperthyroidism or cholestasis. On the other hand, monitoring sarcoidosis obviates the measurement of ACE activity in other biological fluids, e.g., broncho-alveolar and cerebrospinal fluids, in the search of a locoregional dissemination or dis-simulation of the disease. Decreased SACE has been reported in vascular pathologies involving an endothelial abnormality, e.g., deep vein thrombosis, and in endothelium dysfunctions related to the toxicity of chemo- and radiotherapy used in cancers, leukemias, and hematopoietic or organ transplantations. SACE is also of interest for monitoring arterial hypertension treated with specific synthetic ACE inhibitors. These various reasons for determining ACE activity have led to the development of numerous methods. The most widely used is the spectrophotometric assay using hippuryl-histidyl-leucine as substrate. Fluorimetric and radiochemical assays using both classic and novel substrates have been proposed, but they are time consuming, require special apparatus, and are not suited to automation. Kinetic spectrophotometry of furylacryloyl-phenylalanyl-glycyl-glycine hydrolysis is now used extensively because it is easy to automatize. Efforts are now required to standardize one or more of these assays. Indeed, "normal" plasma values differ not only according to the substrate, but also to the method of determination and to sex and age.

A reliable radiometric assay for the determination of angiotensin I-converting enzyme activity in urine.

We present a radiometric assay for the determination of urinary angiotensin-converting enzyme activity, using benzoyl-[1-14C]glycyl-L-histidyl-L-leucine as the substrate. An optimal pH of 8.3, an optimal chloride concentration of 0.375 mol/l and complete inhibition by EDTA-Na2, captopril and enalaprilat confirm the specificity of the assay. Comparison of dialysis and ultrafiltration for concentration of urine showed the existence of angiotensin-converting enzyme inhibitors in human urine. Dialysis against water was the more effective method for avoiding enzyme inhibition. After dialysis of urine, the assay was linear with time and with enzyme concentration; it was highly sensitive (60 mU/l) and showed good reproducibility. Under our technical conditions, we found angiotensin-converting enzyme activity in urine samples with quantitatively abnormal protein contents, but not in normal urine. Urinary angiotensin-converting enzyme did not correlate with proteinuria nor with water-salt parameters or creatinine. We confirm the kidney tubular epithelial origin of the enzyme, and propose the use of our assay to study urinary angiotensin-converting enzyme as a marker of renal tubular damage.

Serum angiotensin-converting enzyme in healthy and sarcoidotic children: comparison with the reference interval for adults.

Angiotensin-converting enzyme (ACE) was measured in serum of 187 healthy children between the ages six months and 18 years. Results were pooled for five-year age intervals and compared with the reference values for adults that we previously determined [Clin Chem 1986;32:884-6). Results for each age group were also studied as a function of sex. Children had higher ACE activities in serum than did adults (P less than 0.001), but these activities were age-related only from age four to 18 years. Adolescents showed sex-related differences, with higher serum ACE activities in boys than in girls (P less than 0.05). Both sex- and age-related differences may be related to a steroid hormonal regulation of ACE biosynthesis. We also verified that children with sarcoidosis (n = 20) had significantly increased serum ACE activity. Such physiological variations in serum ACE activity must be taken into account for diagnosing sarcoidosis in children, for following the course of the disease, and for evaluating the accuracy of therapy.

Clinical reliability of measured and calculated oxygen parameters in surgical patients: influence of hyperventilation.

The acid-base and oxygen status were assessed in patients undergoing abdominal surgery. We determined the measured and calculated parameters described by Siggaard-Andersen in arterial and mixed venous blood before, during and after a controlled hyperventilation. In these dynamic conditions, the mixed venous calculated p50 showed the most interesting variations. Derived values in arterial blood were unreliable since: 1) fitting to hyperbolic tangent function is not possible when SaO2 greater than 0.98; 2) in these patients, the arterio-venous difference in CtO2 is different from 2.3 mmol/L. The acute changes in calculated p50 are in agreement with the effect of hyperventilation on oxygen status.