Oxidized HDL are much less cytotoxic to lymphoblastoid cells than oxidized LDL.

The possible effect of oxidized HDL was investigated on lymphoblastoid cells, in comparison to the cytotoxic effect of oxidized LDL. Oxidation of HDL was promoted by UV-C irradiation, or by copper ion (5 microM) or the combination of the two treatments. HDL extensively treated by UV-C for 20 h did not exhibit any cytotoxic effect on cultured lymphoblastoid cells even at a concentration of 500 micrograms apolipoprotein A-I/ml. In contrast to UV-treated (2 h) LDL, which were highly cytotoxic (already at a concentration of 100 micrograms apolipoprotein B/ml), HDL treated by copper or copper + UV were oxidized, as shown by TBARS formation and PUFA content decrease, but were slightly cytotoxic.

Phospholipid hydrolysis of mildly oxidized LDL reduces their cytotoxicity to cultured endothelial cells. Potential protective role against atherogenesis.

Oxidized low density lipoproteins (LDL) are cytotoxic to cultured endothelial cells and thereby are potentially involved in endothelial cell injury and atherogenesis. Oxidized phospholipids of oxLDL undergo spontaneous hydrolysis (PL-hydrolysis) by LDL-associated phospholipase A2 (PLA2) activities. The present study aimed to investigate whether hydrolysis of oxidized phospholipids contained in mildly oxLDL could influence their cytotoxicity to cultured endothelial cells. PL-hydrolysis (spontaneous or mediated by exogenous PLA2) of mildly oxLDL elicited a significant reduction of their cytotoxicity to cultured endothelial cells. The reduced cytotoxicity of PL-hydrolysed oxLDL was not due to their reduced uptake by cells, but rather to their reduced content of oxidation products which are liberated by PL-hydrolysis and released (at least the more polar compounds) in the aqueous phase, as shown by ultrafiltration experiments. Oxidation products released in the aqueous phase were not or only slightly cytotoxic to endothelial cells, probably because a selective uptake of non oxidized fatty acids as shown by studies of uptake of oxidized and non oxidized [1-14C]linoleic acid. These data suggest that during PL-hydrolysis of mildly oxLDL, (i) oxidized phospholipids are hydrolysed; (ii) oxidation products liberated from oxLDL particles are released (at least in part) to the aqueous phase; (iii) the cytotoxicity of oxLDL to endothelial cells is reduced, probably because oxidized free fatty acids (released by PL-hydrolysis towards the aqueous phase) are not taken up by the cells. Finally, the possibility of a favourable role of PL-hydrolysis of oxLDL against atherogenesis is discussed.

Reduced Na(+)-K(+)-ATPase activity and plasma lysophosphatidylcholine concentrations in diabetic patients.

A fraction from normal human plasma inhibiting Na(+)-K(+)-ATPase has been recently identified as lysophosphatidylcholine (LPC). The aim of this study was to investigate the existence of a relationship between the activity of the cellular membrane Na(+)-K(+)-ATPase and plasma LPC in human diabetes. We studied 10 patients with insulin-dependent-diabetes mellitus (IDDM), 14 patients with non-insulin-dependent diabetes mellitus (NIDDM), and 10 sex- and age-matched control subjects. Plasma LPC concentrations were increased in both IDDM and NIDDM patients compared with control subjects. Na(+)-K(+)-ATPase activity was reduced in both groups of patients in erythrocyte and platelet membranes. There was a significant correlation between the concentrations of plasma LPC and Na(+)-K(+)-ATPase activity in both erythrocyte and platelet membranes (P < 0.01). To investigate the effect of LPC on the enzyme, Na(+)-K(+)-ATPase activity was determined in erythrocyte membranes obtained from six healthy subjects after in vitro incubation with increasing concentrations of LPC (1-10 microM). Enzymatic activity was significantly reduced by in vitro LPC at a concentration of 2.5 microM, with a further decrease at 5 microM. These data suggest that the decrease in Na(+)-K(+)-ATPase activity in diabetes might be due to increased LPC concentrations.

Cerebral circulatory effects of a dopaminergic agonist (apomorphine) in the dog.

Dopaminergic influence upon cerebral circulation was studied in 12 adult mongrel dogs, local cerebral blood flow (lCBF) being measured by the microspheres method. The intravenous injection of apomorphine (0.1 mg/kg) produced heterogeneous effects on lCBF; an increase in lCBF reaching the level of statistical significance was observed in frontal and sensory motor cortices, whereas blood flow was not significantly altered in the other regions examined. When the animals are considered individually, a widespread response was generally observed. Local cerebral blood flow increased from 16% to 85% (depending on the regions examined) in 4 animals, and tended to decrease (particularly in the caudate nucleus) in the 8 others. These opposite effects cannot be related with changes in general metabolic or hemodynamic parameters (PaCO2, PaO2, pH, and arterial pressure). It is thus concluded that the effect of dopaminergic stimulation upon cerebral circulation is more complex than previously reported. These effects could only be partly mediated via vascular receptors and could be the result of modifications of the local metabolic level.

Is cutaneous apoprotein B a better discriminator than serum lipoproteins for atherosclerosis?

Serum lipids, skin apoprotein B (apo B) and skin cholesterol measurements have been investigated in 2 populations: one with normal coronarography, the other with pathological coronarography. Within these 2 populations there were highly significant differences in serum apo B (P less than 0.001), skin cholesterol (P less than 0.01) and skin apo B (P less than 0.001) levels. Skin apo B is a valuable test because its increase is closely related to the coronary heart disease. From these 2 populations, 2 groups with normal serum apo B (less than 1.3 g/l) were selected and compared. No significant differences in the various serum lipids were observed except for triglycerides (P less than 0.05) and serum apo B (P less than 0.05). However, a very significant difference was noticed in the skin apo B (P less than 0.001). With this cutaneous apo B determination it was possible to foresee coronary heart disease in 75% of patients. This test can be therefore considered useful to predict coronary status.

Desialylated low density lipoproteins and atherosclerosis.

Oxidative modification of LDL is accompanied by a number of compositional and structural changes, now well known. In addition, other atherogenic modifications of LDL exist, such as desialylation. The present article summarizes the recent data related to desialylated LDL and to the presence of these LDL in blood plasma of patients with coronary atherosclerosis. In addition, this review examines the sensitivity of these LDL to peroxidative stress.

The diagnostic value of acetylcholinesterase/butyrylcholinesterase ratio in Hirschsprung's disease.

Aganglionosis of large bowel (Hirschsprung's disease; HD) is associated with higher acetylcholinesterase activity (AChE activity). Occasionally, especially in the neonatal period, the AChE activity may not be of diagnostic value. The authors previously reported that simultaneous estimation of butyrylcholinesterase activity (BChE activity) and the determination of AChE/BChE ratio may have discriminatory diagnostic value. They extended this finding to 31 cases of HD, in 16 of which resected tissue was available for study. All cases had histologic confirmation of aganglionosis. The AChE/BChE ratio was found to be higher than 2.0, with the exception of a case in which the biopsy weight was low (i.e., less than 3 mg), even when the AChE activity was normal or borderline. The estimation of AChE/BChE ratio is easy, rapid, and, in the author's experience, of discriminatory diagnostic value.

Increased susceptibility to lipid oxidation of low-density lipoproteins and erythrocyte membranes from diabetic patients.

The aim of the present study was to determine if low-density lipoproteins (LDLs) and red blood cell (RBC) membranes from diabetic patients present an increased susceptibility to lipoperoxidation, which might be related to the increased incidence of atherosclerosis in diabetes. LDLs and RBC membranes were isolated from 11 insulin-dependent (IDDM) and 18 non-insulin-dependent diabetic (NIDDM) patients and exposed to a peroxidative stress by incubation with phenylhydrazine. The susceptibility to peroxidation was determined by measuring the production of thiobarbituric acid-reactive substances (TBARS) after the incubation. The following parameters were also evaluated: plasma glucose, triglycerides (TG), phospholipids (PL), total and high-density lipoprotein (HDL) cholesterol, apolipoprotein (apo) A-I, apo B, hemoglobin A1c (HbA1c), LDL PL and cholesterol, LDL fatty acid composition, and RBC membrane PL and cholesterol. Although they were apparently normolipidemic, diabetic patients showed an increased susceptibility to peroxidation in LDLs and erythrocyte membranes as compared with control subjects. The amount of arachidonic acid in LDLs and the PL concentration of RBC membranes from diabetic patients were significantly higher than in normal subjects. The increased lipoperoxidability of both RBC membranes and LDLs might play a central role in the pathogenesis of the vascular complications of diabetes mellitus.

Role and use of expert systems within the clinical laboratory.

Artificial intelligence can be used, mainly in the form of knowledge based systems, for different parts of clinical laboratory activity. Most of the iterative tasks for control and validation, or for maintenance or repair of automated instruments can be assisted by specialized software. There are now specific programmes (i) for control and validation of the request, where the goal is to modify the requesting behaviour of the physician, using protocol driven requests and immediate feedback, incorporating simultaneously a check of test request redundancy, (ii) for helping in equipment trouble-shooting, (iii) for technical and biological validation because too many QC data are issued from large equipment to be checked and used properly by the technologist whilst the pathologist can be assisted for the final revision of reports and (iv) for assistance in the interpretation of laboratory reports, one of the most valuable aspects of the pathologist's participation in the patient care process.

[Glycoproteins of pleural effusions (author's transl)]. / Les glycoprotéines des liquides pleuraux.

Using single radial immunodiffusion, ten glycoproteins from non purulent pleural fluids have been estimated in different diseases. For five proteins (prealbumin, ceruloplasmin, alpha2HS-glycoprotein, transferrin, beta2-glycoprotein 1) the results have been found not to correlate with the causal disease. However for orosomucoid, alpha1-antitrypsin, haptoglobin, alpha2-macroglobulin and hemopexin, there was good correlation between proteins levels and aetiology. The glycoprotein concentration was low in mechanical effusions from cirrhosis and chronic cardiac failure. It was high in inflammatory, post-embolism and particularly neoplastic effusions. A raised orosomucoid level occurred as the most characteristic of cancer states especially when associated with a parallel increase of the four other glycoproteins. A simultaneously elevated level of these five pleural glycoproteins seems to be a good and significant biological sign for neoplastic effusion diagnosis.

Cholinesterase activities in resected bowel specimens from children with Hirschsprung's disease.

We have measured acetylcholinesterase (AChE) and the butyrylcholinesterase (BChE) activities along strips of resected bowel specimens from thirteen children with Hirschsprung's disease. The AChE activity was variable along the length of the resected bowel, while the BChE activity remained constant. By contrast, the AChE/BChE ratio decreased rapidly to low levels when ganglion cells were present. We suggest that this ratio can be used to determine the normal segments of the bowel, to specify the anatomic forms (i.e. rectal or rectosigmoid) and may be useful in determining the anastomotic level.

Laboratory medicine in France. A jeopardized situation.

The expenses for health care in France have risen considerably during the present decade, ranking third after USA and Canada in the Western world. In spite of the very low cost of laboratory medicine (2.4% of the total expenditure in 1995), clinical laboratories have undergone a severe squeeze, due to two limiting factors; a decrease in the ordering of laboratory tests from private physicians and a reduction in the total expenses for laboratory services from the Social Security. Consequently, there has been unemployment of technical and secretarial staff and severe restriction in investment for buying new equipment. However, hospital laboratories will manage to assume their challenge in developing robotics, automation, molecular pathology techniques and expert systems. Private laboratories, in spite of their efforts to follow the technological advances in automation, will survive thanks to consolidation of regional networks that operate in a cooperative rather than competitive mode. Therefore, the challenge will be not in the adaptation of clinical laboratories, but in the limitation of overspending at the national level and in modification of the behaviour of irresponsible citizens accustomed to spending freely on health care services.

Effect of nicotine and cotinine on the susceptibility to in vitro oxidation of LDL in healthy non smokers and smokers.

Cigarette smoke of which the major component is nicotine plays an important role in the development of cardiovascular diseases. To study the effect of in vitro incubation of LDL with nicotine and its metabolite, cotinine on a copper-induced peroxidation, we monitored the formation of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances production. The LDL studied were taken from six non-smokers (aged 41.5 years) and six smokers who consumed at least ten cigarettes per day (40.7 years). LDL oxidation with CuSO4 showed that cigarette smoking promotes LDL susceptibility to peroxidative modification. During the peroxidation of LDL with nicotine (O to 5 mmol/1) and CuSO4 (5 micromol/l), the formation of hydroperoxides decreased when nicotine concentrations increased and the production of TBARS increased in a concomitant manner. The results showed that the presence of nicotine destabilized the production of hydroperoxides in LDL and increased the formation of secondary oxidation products. On the other hand, cotinine had no effect on LDL oxidative susceptibility in smokers and non-smokers.

Automated technical validation--a real time expert system for decision support.

Dealing daily with various machines and various control specimens provides a lot of data that cannot be processed manually. In order to help decision-making we wrote specific software coping with the traditional QC, with patient data (mean of normals, delta check) and with criteria related to the analytical equipment (flags and alarms). Four machines (3 Ektachem 700 and 1 Hitachi 911) analysing 25 common chemical tests are controlled. Every day, three different control specimens and one more once a week (regional survey) are run on the various pieces of equipment. The data are collected on a 486 microcomputer connected to the central computer. For every parameter the standard deviation is compared with the published acceptable limits and the Westgard's rules are computed. The mean of normals is continuously monitored. The final decision induces either an alarm sound and the print-out of the cause of rejection or, if no alarms happen, the daily print-out of recorded data, with or without the Levey Jennings graphs.

Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies.

In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.

Quantitation of homocysteine in human plasma by capillary electrophoresis and laser-induced fluorescence detection.

Homocysteine (Hcy) represents a branching point between the transsulfuration and transmethylation pathway of methionine. A large increase of plasma concentration of Hcy is observed in patients with inherited hyperhomocysteinemia. A moderated increase (above 10 microM) is also observed in various pathological conditions, such as arterial occlusion, hypertension, hyperlipidemia and chronic renal failure. While amino acids were largely studied using capillary electrophoresis with UV or laser-induced fluorescence detection (LIF), thiol-amino acids were not. In this work we present a new approach for testing homocysteine in human plasma using CE-LIF and fluorescein isothiocyanate. The low fluorescence yield of the fluorescein thiocarbamyl (FTC) thiol-amino acids limits, probably, the sensitivity of the detection to 8 x 10(-10) M (instead of 10(-12) M for FTC-arginine).

A data management program for the Electra 800 automatic analyser.

The Electra 800 automatic coagulation analyser rapidly performs most chronometric coagulation tests with high precision. To facilitate data handling, software, adaptable to any PC running under MS-DOS, was written to manage the analyser. Data are automatically collected via the RS232 interface or can be manually input. The software can handle 64 different analyses, all entirely 'user defined'. An 'electronic worksheet' presents the results in pages of ten patients. This enables the operator to assess the data and to perform verifications or complementary tests if necessary. All results outside a predetermined range can be flagged and results can be deleted, modified or added. A patient's previous files can be recalled as the data are archived at the end of the day. A 120 Mb disk can store approximately 130,000 patient files. A daily archive function can print the day's work in alphabetical order. A communication protocol allows connection to a mainframe computer. This program and the user's manual are available on request, free of charge, from the authors.

Lower alkaline phosphatase activity and occurrence of an abnormal hybrid intestinal/tissue non-specific isoform in Down's syndrome amniotic fluids.

Quantitative and qualitative analysis of alkaline phosphatases (AP) was performed on amniotic fluid in 59 normal pregnancies and 14 Down's syndrome (DS) pregnancies at 16, 18 and 19 weeks of gestation. In DS cases, intestinal and placental isoenzyme levels were significantly reduced (P<0.001) and the AP electrophoretic pattern was seen to be modified on polyacrylamide gel electrophoresis. A unique component was detected. After extraction and purification of the abnormal isoenzyme, peptide fragments obtained after cyanogen bromide cleavage indicated a hybrid heterodimeric AP composed of intestinal and tissue non-specific subunits, as evaluated by SDS polyacrylamide gel electrophoresis.

The influence of phthalate esters on human plasma lecithin/cholesterol acyltransferase.

The effect of various phthalate esters on the lecithin/cholesterol acyltransferase activity in man was studied in vitro. The enzymatic activity was strongly reduced with all phthalates except for the dimethyl phthalate. The inhibition rate depends on the phthalate concentration and also on the carbon number of the alkyl groups of phthalates.

Lipoprotein peroxidation in adult psoriatic patients.

Psoriasis, a chronic inflammatory skin disease characterized by an aberration of lipid metabolism, has been associated with an increased risk for atherosclerosis. Since oxidatively modified lipoproteins are involved in the pathogenesis of atherosclerosis, we investigated the lipid composition and in vitro induced peroxidation of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from psoriatic patients. 11 male adult psoriatics and 16 male age-matched healthy subjects were studied. Lipid peroxidation of VLDL and LDL was performed by incubation with CuSO4 for 24 h at 37 degrees C. The compositional analysis showed a significant increase in triglycerides and phospholipids, both in VLDL (p < 0.05) and in LDL (p < 0.001) from psoriatic patients, compared with controls. Moreover a significant increase in total cholesterol (TC) (p < 0.01) and apoprotein (P) (p < 0.05) was found in LDL from psoriatics. The levels of thiobarbituric acid reactive substances (TBARS), as a measurement of lipid peroxidation, were significantly higher in Ox-VLDL and in Ox-LDL from psoriatics (p < 0.01) than the corresponding values in controls. Moreover, basal values of TBARS were significantly higher in VLDL and LDL from psoriatic patients than those from controls. In conclusion, the lipoprotein compositional changes associated with the modifications of TBARS before and after Cu2+ treatment of lipoproteins may suggest an increased risk for atherosclerosis in adult psoriatic patients.