Characterization of lipoproteins during human cholestasis.

We have characterized changes in lipoproteins from cholestatic individuals and reproduced them by incubating lipoproteins from healthy individuals with cholic acid. The cholestatic patients showed an increase in low density lipoprotein (LDL) (>85%), with a smaller proportion of esterified cholesterol, and a fall in high density lipoprotein (HDL) (<10%), with a larger proportion of phospholipids. The protein composition of cholestatic HDL1 was characterized by a smaller proportion of apo A (I, II) and a prominent apo E fraction (39% vs. 9%). These changes involved an increase in degree of molecular packing (order) of HDL1. The addition of cholic acid to serum from healthy individuals altered the lipoprotein distribution, with an increase in LDL, the disappearance of HDL2 and HDL3 and the appearance of HDL1. These HDL1 were characterized by increased phospholipid and reduced apo AI fractions. They also showed a lower density and appeared as spherical particles in contrast to cholestatic HDL. Incubation of healthy HDL with cholic acid in vitro reproduces some of the alteration observed in cholestatic HDL.

Total automated method for determination of cholesterol concentration in high density lipoprotein by heparin-magnesium-albumin precipitation.

We describe an automated procedure for determination of high-density-lipoprotein cholesterol in serum by use of heparin-MgCl2-albumin reagent. 'AutoAnalyzer' II equipment was used in which high-density lipoprotein was separated from precipitate containing very-low and low-density lipoproteins by filtering on-line across a cellulose acetate membrane. Cholesterol concentration was measured by the enzymic method of Allain. Good correlation was obtained using the automated method compared to the heparin-MgCl2-albumin (r=0.99) or sodium phosphotungstate-MgCl2 procedure (r=0.97). Contamination was minimal with a contamination coefficient lower than 2.5%. The method presented was linear (0 to 2.50 mmol/1) and could be applied to lipemic samples with a coefficient of variation better than 3%. This study showed thus that this new procedure was suitable for routine work with a good rate (60 samples/h) in almost any laboratory.

Changes in human high density lipoproteins in patients with extra-hepatic biliary obstruction.

Plasma high density lipoproteins (HDL) from patients with obstruction of the common bile duct were studied by crossed immunoelectrophoresis and isoelectric focusing. All cholestatic HDL fractions were rich in phospholipids (51.5 +/- 9%) with high proportions of free cholesterol (13.8 +/- 1.5%). Moreover, crossed immunoelectrophoresis of sera against anti-Apo A revealed the presence of multiple immunoprecipitates sharply contrasting with the pattern formed by normal sera. Tandem crossed immunoelectrophoresis against anti-Apo A and anti-Apo B was performed with whole serum and with the HDL fraction from various cholestatic subjects. Crossed identity was observed for two of these precipitates, which could be explained by the decrease in HDL stability due to the detergent effect of bile salts. The most noteworthy changes found in cholestatic patients appeared to be the apolipoprotein pattern of HDL. Both Apo AI (48%) and Apo AII (5.5%) were greatly diminished and Apo E was present in remarkably high amounts (39%) with two additional isoforms (Apo E'1 and Apo E'2).

Effects of postprandial hyperlipemia on the vitamin E content of lipoproteins. GERBAP section Lipoprotéines. Groupe d'Evaluation et de Recherche de l'Assistance Publique des Hôpitaux de Paris.

Delayed postprandial clearance of triglyceride-rich lipoproteins (TGRL) could induce a decrease of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) vitamin E content through its transfer into TGRL. We thus studied lipoprotein vitamin E content during postprandial hypertriglyceridemia induced by a high fat meal without vitamin E supplement. Venous blood was drawn from five healthy male volunteers following a 12 h fast at (t0) then 2 h (t2), 4 h (t4), 6 h (t6) and 8 h (t8) after a 80 g fat meal. In plasma, only TG significantly varied during the postprandial period with a large interindividual variability. Mean composition of lipoproteins in terms of mass was not significantly modified. The amount of vitamin E significantly increased in TGRL and decreased in LDL plus HDL at t4 and t6 relative to t0. Vitamin E content of TGRL and LDL but not of HDL decreased significantly at t4. The mean decrease was 20% (range 5%-54%) for the LDL. LDL- and HDL vitamin E content correlated inversely with plasma TGRL levels. Our data suggest that LDL from subjects with delayed chylomicron clearance could be less protected against oxidation.

Differential measurement of monomeric and polymeric IgA by one-directional immunoelectrodiffusion.

A technique to specifically quantify monomeric IgA and total IgA in colostrum has been developed using a modified one-dimensional immunoelectrophoretic assay. This method employed electrophoresis in antibody-containing polyacrylamide-agarose gel in the presence of a gel barrier which blocks polymeric IgA. The addition of PEG (polyethylene-glycol 6000) to the anodic gel increased the sharpness of the peaks, the height of which was proportional to the antigen concentration. This method proved to be sufficiently simple, precise, reproducible (CV less than 3%) and linear (from 20-300 mg/l) to measure the monomeric IgA: total IgA ratio rapidly (14 +/- 4.5% for 20 samples in duplicate). Immunoelectrodiffusion studies confirmed that human colostral and serum IgA standards could be used to determine directly monomeric IgA, total IgA and polymeric IgA levels (by difference) rather than to apply correction factors to estimate these IgA levels.

Type IV hyperlipoproteinemia in patients with algodystrophy.

The present work studies lipid metabolism in patients with algodystrophy (AD). A correct positive correlation (r = 0.75) between the triglyceride levels and low density lipoprotein (LDL)/very low density lipoprotein (VLDL) ratio and the VLDL increase observed by gel disk electrophoresis confirm that a type IV hyperlipoproteinemia is associated with AD. In contrast, the degree of high density lipoprotein (HDL) saturation in cholesterol (HDL-cholesterol/HDL-phospholipids) and the classical atherogenous index (cholesterol/HDL-cholesterol) were not modified. The decrease of plasma post-heparin lipolytic activities (PHLA) was not significant but further studies should be performed to correlate PHLA with a reduced activity of the adipose tissue lipoprotein lipase.

Individual free fatty acids and lactate uptake in the human heart during severe sepsis.

Coronary haemodynamics and myocardial metabolism of nonesterified fatty acids (NEFA) and lactate were studied in 11 patients with severe sepsis, and compared to 10 control subjects. Coronary sinus blood flow was evaluated by thermodilution. Arterial and coronary sinus blood samples were collected for the measurement of lactate and total and individual NEFA concentrations both in septic and control patients. There was an increase in lactate and total NEFA arterial concentrations with a marked increase in palmitic and linolenic acids. The uptake of the main NEFA (C14:0 to C18:2) was significantly decreased. In the control group, individual NEFA uptake was proportional to their arterial concentrations. This relationship was not observed in patients with sepsis: there was no preferential extraction of any particular NEFA. Furthermore, in patients with sepsis, myocardial oxygen consumption did not correlate with NEFA, but only with lactate uptake. Alterations in NEFA uptake were found to be constant during severe sepsis and are consistent with major disturbances in myocardial metabolism.

Free fatty acid microdetermination by gas-liquid chromatography without transmethylating effects of methylation procedure.

A fast and practicable gas-liquid chromatographic method for the quantitation of non-esterified fatty acids (C14:0-C18:2) from 100 microliter plasma is described. This technique includes extraction, purification using the solvents n-heptane and 0.5 M Na2CO3, and methylation by 0.1 M HCl-methanol. Extraction, quantification and optimal methylation conditions without transmethylation have been investigated. Reproducibility is good and results agree with values found in the literature.

[Serum phospholipids determination by an automatic enzymatic method (author's transl)]. / Dosage des phospholipides sériques par méthode enzymatique appliquée au flux continu.

The serum phospholipids determination by an automatic enzymatic method (AEM) is suggested in this study. A comparison was made between the classical chemical method, the manual enzymatic method (MEM) and the AEM one. 100 serum samples were tested in this experiment and the correlation coefficient between these methods was found to be acceptable. The result indicates a good precision and a negligible contamination. The calibration curve is linear to 5 mmol/l. Triglyceride and cholesterol concentrations, glucose and high concentrated uric acid presence do not interfere on the value measured. However, the haemolysis and a large quantity of bilirubin in sera tend to induce a variation in the phospholipids determination.

[Estimation of serum lipids: method without extraction adapted to autoanalysis (author's transl)]. / Dosage des lipides sériques: méthode sans extraction adaptée à l'autoanalyseur.

An entirely automatic method, without prior extraction, is described for the determination of total serum lipids by a nephelemetric method. The results are compared with those obtained by Canal's method. There is a good correlation between the two technics. With a similar concentration of lipid the respective quantities of cholesterol and triglyceride do not intervene in the flocculation. The respective influence of the bilirubin concentration, hemolysis, and the concentration and nature of the serum proteins is considered. This method should permit simultaneous autoanalysis of the three parameters of classical lipid investigation: total lipids, triglycerides and cholesterol.

[Enzyme-immuno assay for total estrogens and human placental lactogen. Comparison with radio-immuno-assay in normal pregnancy-monitoring (author's transl)]. / Dosages sériques immuno-enzymatiques des estrogènes totaux et de l'hormone lactogène placentaire. Comparaison avec la méthode radio-immunologique dans la surveillance de la grossesse normale.

The concentrations of estrogens (E) and human placental lactogen (HLP) are estimated in sera by radio immuno-assay (RIA) and enzyme-immuno-assay (EIA). Statistical data indicate mean intra-assay variation coefficients of 7% and 12% for E and HLP tests, respectively. The correlation coefficient (RIA/EIA) are found higher than 0,9% for both hormonal assays. The dilution curves obtained by RIA and EIA are similar. However, Student'test gives a significant difference for E determination. In fact, total E and E 3 only are measured by EIA and RIA, respectively. In most cases biological interferences are negligible except for HLP in presence of higher protein or haemoglobin levels. RIA and EIA are performed to study serum HLP and E levels throughout normal pregnancies. Results allow to use EIA for HLP and E evaluations in pregnancy-monitoring.

[Comparison of Orth-Evac and Solcotrans Plus devices for the autotransfusion of blood drained after total knee joint arthroplasty]. / Comparaison des dispositifs Orth-Evac et Solcotrans Plus pour l'autotransfusion du sang drainé après arthroplastie totale du genou.

Surgical wound blood which is ched through drains after total knee replacement surgery with a tourniquet may be returned to the patient using special collecting devices. This study aimed to compare two systems, Orth-Evac and Solcotrans Plus an to assess the safety of the reinfusion of non washed blood cells. It included 30 patients scheduled for total knee replacement surgery, free from tumoral or coagulation disease and allocated randomly in three groups of 10 each: the Orth-Evac group (OGr), the Solcotrans Plus group (SGr) and the Control group (CGr). The devices, not containing an anticoagulant, were connected to the deep suction drains in the operating room, after skin closure and before the tourniquet removal. The salvaged blood was reinfused in the subsequent six hours via a 40 microns filter. The volume of collected blood was measured and homologous blood was added as required, to maintain a hematocrit of 30%. A blood sample was obtained the day before surgery (D - 1), before reinfusion (D0), two hours later (D + 2h), one day later (D + 1), and from the collecting device before reinfusion. The statistical analysis used the Kruskal-Wallis test and Steel-Dwass procedure to confirm the difference between two groups. The three groups did not differ in age, weight, height and gender. The volume of salvaged and autotransfused blood was 925 +/- 156 mL in OGr and 605 +/- 178 mL in SGr respectively, transfusion of homologous blood was required in two patients of OGr, four of SGr and six of CGr. At D + 1, the hematocrit was comparable in all groups (OGr = 28%, SGr = 28.2% and CGr = 28.5%).(ABSTRACT TRUNCATED AT 250 WORDS)

[Oral administration of sodium citrate before general anesthesia in elective cesarean section. Effect on pH and gastric volume]. / Ingestion de citrate de sodium avant l'anesthésie générale pour césarienne programmée. Effet sur le pH et le volume du contenu gastrique.

A study was carried out to assess the effects of oral sodium citrate on the pH and volume of gastric content in 52 unpremedicated women undergoing elective Caesarean section under general anaesthesia. The patients were randomly assigned to two groups: group T (n = 26) without antacids and group C (n = 26) receiving orally 30 ml of 0.3 M sodium citrate solution 5 min before induction of anaesthesia. After tracheal intubation, a size 18 double lumen naso-gastric tube was introduced. The gastric content was sampled 10 min (n = 52), 50 min (n = 52) and 80 min (n = 24) later. Before extubation, the stomach was completely emptied and the naso-gastric tube removed. On each sampling time, the pH was measured with a digital pHmeter; a search for biliary salts (pyloric reflux) was carried out with thin layer chromatography; the volume of the gastric content was measured using the phenol red dilution method. At 10 min, none patient in group C had a gastric pH less than 2.5, in the opposite to 92% of patients in the control group T. The alkalinizing effect of the citrate lasted throughout the evaluation time. The mean pH was respectively 5.3 +/- 1.07, 5.23 +/- 1.2 and 5.53 +/- 1.3 at 10, 50 and 80 min in group C (controls: 1.9 +/- 0.7; 2.49 +/- 1.3; 2.40 +/- 0.61). However, the citrate solution increased significantly the volume of the gastric content. It was respectively 51.1 +/- 29.9 ml, 79.15 +/- 70.9 ml and 32.9 +/- 22.1 ml; 29.6 +/- 28.5 ml; 26.6 +/- 10.9 ml).(ABSTRACT TRUNCATED AT 250 WORDS)

[Critical aspects of actual methods for high-density-lipoprotein cholesterol quantitation]. / Aspects critiques des méthodes actuelles de détermination du cholestérol-HDL.

In this study we compare various methods for h;igh-density-lipoprotein cholesterol quantitation: ultracentrifugation; electrophoresis either on cellulose acetate or on agarose gel; precipitation methods with polyanions-bivalent cations; adsorption procedure using concanavalin A (Con A). quantification by electrophoresis is slow and not precise. Except heparin-Mn++ and dextran (MM 5 X 10(5) and 2 X 10(6)) - CaCl2 procedures, precipitation and Con A methods own a good repeatability (VC 2%) and give a linear response after VLDL or LDL-VLDL addition to tested sera. Regression analysis of the data obtained using these methods in pairs demonstrate a correct accuracy (r > 0.95). The Student t-test shows that the usual values must be evaluated for each method. We suggest to use either phosphotungstate - Mg++ or Con A procedure as method for HDL cholesterol quantitation.

Heparin--magnesium chloride--albumin method for enzymic measurement of cholesterol in high-density lipoprotein.

We describe a new method for measuring high-density-lipoprotein cholesterol in sera, with use of heparin, MgCl2, and albumin to precipitate very-low- and low-density lipoproteins, Lipoprotein electrophoresis and immunochemical methods show that high-density lipoprotein is completely and specifically isolated in the supernate, the cholesterol concentration of which is estimated by the enzymic method of Allain et al. (Clin. Chem. 20, 470, 1974). The precision of the method is excellent (CV = 1%) and the response is linear. Values so obtained correlate well with those obtained with the heparin-Mn2+ (r = 0.98) or the phosphotungstate-Mg2+ (r = 0.98) procedures. The technique is appropriate for routine clinical laboratory use.

[Serum amylase determination : automated continuous flow method (author's transl)]. / Mesure de l'amylase sérique : méthode automatisée en flux continu.

A simple automated assay was described for the determination of serum amylase using a chromogenic substrate. The reaction was stopped by filtration across a cellulose nitrate membrane. The analytic parameters of this method were studied. Repeatability, reproducibility and precision were convenient. The calibration curve was linear up to 850 U/I. An excellent correlation was obtained after comparison with the manual method (Phadebas amylase test). The effects of haemolysis and very high ascorbic concentrations of bilirubin, glucose, uric acid and ascorbic acid were negligible on the value measured. The serum turbidity was alone to induce a variation by excess.

[Metabolism and septic shock]. / Métabolisme et choc septique.

Despite continuous monitoring progress, septic shock remains the main cause of death in Intensive Care Units. Septic stress triggers a complex array of biological modifications resulting in an acquired disease of intermediary metabolism whose main characteristic is an inefficient cellular use of glucidic, lipidic and protidic substrates together with oxygen. The purpose of this review is to present metabolic disturbances observed during sepsis and septic shock.

Alpha-1-acid glycoprotein concentrations and protein binding of propranolol in Sprague-Dawley and Dark Agouti rat strains treated by phenobarbital.

Propranolol binding to rat serum samples was studied after 7 days of phenobarbital (PB) administration to induce alpha-1-acid glycoprotein (AAG). In male Sprague-Dawley rats, serum AAG concentrations and the bound/free propranolol ratio (B/F) were increased slightly after enzyme-inducing drug administration (mean +/- S.D.): from 0.30 +/- 0.04 to 0.49 +/- 0.05 g/l for AAG and from 6.79 +/- 1.24 to 8.81 to 1.31 for B/F. In male DA (Dark Agouti) rats, the same parameters increased greatly: from 0.20 +/- 0.01 to 1.38 +/- 0.13 g/l for AAF and from 8.75 +/- 1.01 to 27.15 +/- 3.28 for B/F. PB concentrations determined on the same serum samples were similar in both rat strains. Female DA rats also were responsive to PB administration. Propranolol binding to DA rat serum from the treated group was characterized by an increase in high-affinity binding site concentrations. The binding of propranolol to isolated rat AAG at various concentrations (0.2-1 g/l), alone or associated with rat serum albumin at a constant concentration (36 g/l), also was studied. Propranolol binding capacities to rat AAG were increased 2.4 times by the presence of albumin and consequently B/F values for both proteins agreed with values obtained with serum samples. These results indicate that increased propranolol binding to rat serum induced by PB administration is probably a direct consequence of increased concentrations of AAG. The high sensitivity of DA rats to PB suggests that this strain should be used preferentially for the study of the effect of cytochrome P-450 inducers on AAG metabolism and cationic drug binding.

The immunomodulating agent RU 41740 complexed with very-low-density lipoproteins enhances human polymorphonuclear neutrophil oxidative metabolism in vitro.

Added to human serum in vitro, RU 41740, an immunomodulating agent extracted from Klebsiella pneumoniae, binds selectively to lipoproteins containing apolipoprotein B (low-density lipoproteins and very-low-density lipoproteins, VLDL) and, at higher concentrations, to lipoproteins containing apolipoprotein A (high-density lipoproteins). The fact that lipoproteins modulate polymorphonuclear neutrophil (PMN) functions led us to suspect that the VLDL-RU 41740 complex might affect PMN functions. In this study, the effect of this complex on PMN superoxide generation was measured in the presence and absence of the classical stimulants formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate. The VLDL-RU 41740 complex enhanced the stimulating effect of VLDL on quiescent PMN, but not following stimulation with formyl-methionyl-leucyl-phenylalanine. In contrast, it partially counteracted the inhibiting effect exerted by VLDL alone on phorbol myristate acetate stimulation. Such a complex might be formed in vivo during RU 41740 therapy and constitute an important feature in the immunostimulating properties of the drug.