Influence of alpha-1 glycoprotein acid concentrations and variants on atazanavir pharmacokinetics in HIV-infected patients included in the ANRS 107 trial.

Atazanavir is an HIV-1 protease inhibitor with high protein binding in human plasma. The objectives were first to determine the in vitro binding characteristics of atazanavir and second to evaluate whether plasma protein binding to albumin and alpha-1 glycoprotein acid (AAG) influences the pharmacokinetics of atazanavir in HIV-infected patients. For the in vitro study, atazanavir protein binding characteristics were determined in AAG- and albumin-containing purified solutions. Atazanavir was found to bind AAG on a high-affinity saturable site (association constant, 4.61x10(5) liters/mol) and albumin on a low-affinity nonsaturable site. For the in vivo study, blood samples from 51 patients included in trial ANRS 107--Puzzle 2 were drawn prior to drug intake at week 6. For 10 patients included in the pharmacokinetic substudy, five additional blood samples were collected during one dosing interval at week 6. Atazanavir concentrations were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Albumin concentrations, AAG concentrations, and phenotypes were also measured in these patients. Concentrations of atazanavir were modeled using a population approach. A one-compartment model with first-order absorption and elimination best described atazanavir pharmacokinetics. Atazanavir pharmacokinetic parameters and their interindividual variabilities were as follows: absorption rate constant (ka), 0.73 h(-1) (139.3%); apparent clearance (CL/F), 13.3 liters/h (26.7%); and apparent volume of distribution (V/F), 79.7 liters (27.0%). Atazanavir CL/F decreased significantly when alanine aminotransferase and/or AAG levels increased (P<0.01). The ORM1*S phenotype also significantly increased atazanavir V/F (P<0.05). These in vivo results indicate that atazanavir pharmacokinetics is moderately influenced by its protein binding, especially to AAG, without expected clinical consequences.

[Multicenter evaluation of four homogenous LDL-cholesterol assays]. / Evaluation multicentrique de quatre méthodes de dosage direct du cholestérol-LDL.

International guidelines emphasize the importance of LDL cholesterol (LDL-C) assay in the care and follow-up of patients with cardiovascular risk. Most studies and common practice use Friedewald's formula for LDL-C calculation. The accuracy of the result depends closely on the precision of the input parameters (total cholesterol, triglycerides (TG) and HDL cholesterol), and discrepancies between calculated LDL-C and measurement by reference methods appear when TG exceed 4.5 mmol/L, or in the presence of abnormal lipoproteins. These restrictions and uncertainties in calculations have prompted the recent development of direct and homogeneous methods that fit all analyzers. A multicenter evaluation of four direct assays of LDL-C (Daiichi, Denka Seiken, Kyowa, Wako) was carried out on 45 serum samples (TG below 3.1 mmol/L) in eight laboratories using different analyzers. For three methods (Daiichi, Kyowa, Wako), the interlaboratory reproducibility was markedly improved relative to that of calculation. A strong correlation was found for all new methods when compared with a beta-quantification assay. Average bias in Denka Seiken assays was greater than Kyowa's and Daiichi's (although less dispersed for the latter) and for Wako all bias were positive. The relationship between bias variations and the lipid parameters of the samples was studied. Three methods, Daiichi, Kyowa and Wako, revealed a significant positive correlation between bias and serum VLDL-C/TG ratio, clearly indicating that cholesterol enrichment of VLDL was a source of variability in these assays. Specificity of the four methods was tested in situation of dyslipidemia by spiking isolated lipoproteins (chylomicrons, VLDL and HDL). This experiment revealed differences in behavior, most evidently upon addition of VLDL. No method was truly specific, but up to 8 mmol/L of TG the variations were acceptable. In the presence of type III hyperlipoproteinemia, however, only the Denka Seiken method was reliable. Linearity up to 20 mmol/L (Daiichi, Denka Seiken) or 14 mmol/L (Kyowa, Wako) of LDL-C allows these tests to be used in main routine cases. New direct assays are an obvious technological advance in terms of analytical performance and conveniency. Their use for the diagnosis and follow-up of hyperlipidemic patients offers an alternative that overcomes the limitations of the Friedewald calculation.

Interleukin-1 beta and interleukin-6 stimulate 2-methylaminoisobutyric acid uptake in HepG2 cells.

The metabolic response to inflammation involves an increased uptake of amino acids in the liver. It has been suggested that cytokines, such as interleukin-1 beta and interleukin-6, could be involved in this increased amino acid uptake. We investigated the role of these two inflammatory cytokines in regulating hepatic amino acid transport systems in the human hepatoma cell line, HepG2. Uptake of methylaminoisobutyric acid, the most specific known substrate of system A, and of glutamine, both transported by other sodium-dependent transport systems ASC and N, was assayed after incubation of the cells for various times with cytokines, using the cluster-tray method. Interleukin-1 beta and interleukin-6 (1000 U/ml) stimulated methylaminoisobutyric acid uptake by 36 +/- 6 and 41 +/- 4%, respectively (per cent +/- SD, n > or = 6). Under our experimental conditions, these cytokines had no effect on glutamine uptake. The stimulatory effect on methylaminoisobutyric acid uptake was not increased by combining the cytokines or by the presence of dexamethasone. The cytokine effect was abolished by cycloheximide, suggesting the involvement of de novo protein synthesis in this activation of transport system A. These data demonstrate that, in our culture conditions, interleukin-1 beta and interleukin-6 indirectly exert a stimulatory effect on methylaminoisobutyric acid transport in HepG2 cells.

Evidence for a bacteriocin-like substance produced by a new strain of Streptococcus sp., inhibitory to gram-positive food-borne pathogens.

A new strain of Streptococcus sp. (CNCM I-841) isolated from a commercial probiotic product was shown to be antagonistic towards several food-borne pathogens including Clostridium sp. and Listeria monocytogenes. This strain produced and excreted an antibacterial substance in MRS broth. The inhibitory substance was different from hydrogen peroxide, since it was unaffected by catalase. It was sensitive to proteolytic enzymes, indicating that the active moiety of the inhibitor was proteinaceous in nature, and it had no effect on its producer strain. These properties suggested that the inhibitory substance could be considered as a bacteriocin-like substance. The antimicrobial substance was also produced in M17 and tryptose broths if they were supplemented with Tween-80. Partial purification allowed a 10.5-fold increase in the specific activity. A preliminary characterization showed that it was active against lactobacilli, Enterococcus faecalis, Clostridium sp. and Listeria sp. It was not affected by 2-h treatment at 60 degrees C, but was sensitive to treatments at 100 degrees C and to autoclaving at 121 degrees C. The activity was not affected by treatments at pH values ranging from 2 to 11.

Circulating cardiac troponin I in trauma patients without cardiac contusion.

OBJECTIVES: To describe the evolution and the diagnostic value of cardiac troponin I (cTnI) and to relate its concentrations with the indicators of injury in trauma patients. DESIGN: Prospective, observational study of 17 young, previously healthy, mechanically-ventilated patients during the early post-traumatic period in the Surgical ICU of a University Hospital. METHODS: Serial measurements of serum cTnI, total creatine kinase activity (CKtot) and its isoenzyme MB (CK-MB) (on admission, 12 h later, then daily for 7 days), clinical data and repeated electrocardiographic (ECG) and transesophageal echocardiographic (TEE) recordings. RESULTS: Rhabdomyolysis was observed in all the patients with a significant relationship between CK-MB and CKtot. Despite the fact that no patient demonstrated ECG or TEE signs of myocardial contusion, elevated serum levels of cTnI were observed in six patients (35%) without obvious dilutional interference. As compared with the others, these patients exhibited a more frequent arterial hypotension (83% vs 18%, p = 0.035), required greater volume expansion on day 1 (22,000 vs 8,500 ml, p = 0.027) and usually demonstrated early (83% vs 9%, p = 0.005) and late (66% vs 9%, p = 0.028) multiple organ dysfunction syndrome. CONCLUSIONS: Taking into account the high reported sensitivity and specificity of cTnI dosage, the present results suggest cTnI can play a role in the evaluation of indirect myocardial injury following traumatic shock.

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.

Effect of cyclosporine A on mercury-induced autoimmune glomerulonephritis in the Brown Norway rat.

To test the effect of cyclosporine A (CsA) in mercuric chloride (HgCl2)-induced nephritis in the Brown-Norway (BN) rat, we treated groups of intoxicated rats with varying doses of CsA for a period of 2 months. All manifestations of HgCl2-induced disease were prevented in rats treated concurrently with CsA at either 7 or 10 mg/kg/day. Partial suppression was evident at lower daily doses, but not with bi-weekly CsA administration. The initial phase of HgCl2-induced nephritis could be completely suppressed with a short, 15 day course of CsA. The later phase of the disease could be tempered by CsA administration starting on day 10 after the first HgCl2 injection. The optimal regimen of 7 mg/kg/day for 60 days was not associated with any evidence of CsA toxicity. CsA appears to interfere with the polyclonal activation of B cells observed in HgCl2-induced autoimmune disease, accounting for its striking preventive and curative effect in this model.

[Circulating troponin I assay: uses and misuses in medical practice]. / Le dosage de la troponine I: intérêt et limites en pratique médicale courante.

Cardiac troponin I (cTnI) contributes to the modulation of the myocardial contractile function in the troponins complex. The assay of circulating cTnI is highly specific of the cardiac isoform and allows a quantification of the myocardial injury provided that both free and combined cTnI were recognized by the antibodies. The assay is a major contribution to the diagnosis of postoperative cardiac complications through its specificity because peripheral muscle trauma is a confounding factor during this period. Circulating cTnI mirrors the magnitude of an acute circulatory failure whatever the origin (cardiac, hemorrhagic or septic). A positive cTnI assay in an "asymptomatic" patient has to be confirmed and needs some investigations to discard an occult cardiac disease; cTnI has a limited role by itself in predicting mortality and hospital admissions. Circulating cTnI confirms the severity of an acute coronary syndrome and has a level-dependent prognostic value.

Interpretation of lipoprotein-associated phospholipase A2 levels is influenced by cardiac disease, comorbidities, extension of atherosclerosis and treatments.

PURPOSE: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory biomarker secreted in the atherosclerotic plaque. Blood levels of Lp-PLA2 predict future cardiovascular events in patients with ischemic disease and heart failure. This association seems to be independent of traditional cardiovascular risk factors. The aims of our study were (1) to assess relationships between Lp-PLA2 levels, cardiac disease and treatments; (2) to evaluate the association of Lp-PLA2 level with the severity of angiographic coronary artery disease (CAD) and the extracoronary atherosclerosis. METHODS: Between December 2009 and June 2010, 494 subjects were recruited from a population scheduled for diagnostic coronary angiography. Routine clinical (age, gender, BMI and treatment), cardiac (echocardiography, coronarography, carotid ultrasonography) and biochemical parameters were recorded for all patients. Lp-PLA2 mass concentration was assessed in serum with a Plac®-test turbidimetric immunoassay. Control Lp-PLA2 values were specifically obtained in 61 healthy subjects aged 44.5 ± 17.6 years (range: 25 to 59 years) without known cardiovascular risk factors (diabetes, smoking, hypertension, dyslipidemia) or cardiac treatment. RESULTS: In healthy controls, mean Lp-PLA2 level was 163 ± 43 µg/L (166 ± 45 µg/L in men and 159 ± 39 µg/L in women, non significant difference). In our cohort of 494 patients (69.8% men) aged 64.2 ± 16.7 years, the main etiologies of cardiomyopathies were ischemic (40%), valvular (22%), cardiac failure with left ventricular (LV) dysfunction (14%), infection (5%) and aortic aneurysm (7%). Mean Lp-PLA2 levels were 216 ± 17 µg/L. Lp-PLA2 correlated with age, BMI, current smoking, history of hypertension but not with diabetes and gender. The bivariate analysis showed a significant correlation between Lp-PLA2, and BMI (p=0.001) but no correlation with serum creatinine or NYHA status. A multivariate correlation showed that Lp-PLA2 was associated with total cholesterol, LDL-cholesterol and apoB (r=0.95, p<0.0001) but not with Lp(a). We observed that Lp-PLA2 was significantly associated with treatments such as statins and ACEi/ARA2 but not with ß-blockers, antiaggregant drugs or diuretics. Lp-PLA2 levels were significantly higher in patients with CAD than in patients without CAD (223 ± 54 vs. 208 ± 52 µg/L, respectively; p<0.007). Moreover, Lp-PLA2 levels were significantly higher in patients with the most extensive angiographic CAD [single (n=24)=215.2 ± 52 µg/L; two (n=55)=222 ± 53 µg/L and three vessels (n=140)=251.9 ± 53.7 µg/L, respectively; p<0.0001]. Patients with heart failure, sepsis or aortic aneurysm had increased Lp-PLA2 levels: 256.2 ± 46.8; 226.7 ± 47.3; 218.1 ± 38.9 µg/L, respectively, as compared to controls (p<0.0001). In patients with carotid artery disease, Lp-PLA2 significantly increased with the severity of atherosclerosis. Mean Lp-PLA2 levels were 218.8 ± 51 µg/L in the group without any stenosis (n=108), 224 ± 51 µg/L in the group with mild stenosis (n=101), and 231 ± 46 µg/L in the group with severe stenosis (n=22); p=0.004. CONCLUSION: This study clearly shows that interpretation of Lp-PLA2 levels needs a good assessment of cardiac parameters and treatments, especially statins and ACEi/ARA2. Lp-PLA2 levels are significantly associated with coronary heart disease and with the extension of extra coronary disease after adjustment for age and gender.

Manganese superoxide dismutase dimorphism relationship with severity and prognosis in cardiogenic shock due to dilated cardiomyopathy.

The aim was to determine (a) Ala-16Val-SOD2 dimorphisms; (b) allelic frequency and phenotype of a common Pro-Leu polymorphism in GPx1, in a cohort of patients with a cardiogenic shock (CS) due to dilated cardiomyopathy without acute coronary syndrome. Consecutive patients with de novo CS that worsened a dilated (DCM) or ischemic (ICM) cardiomyopathy. Congenital heart disease, pacemaker and other shock aetiologies were excluded. To determine oxidative stress (OS), this study evaluated lipid peroxidation, protein oxidation and erythrocyte GPx, SOD and catalase activities. Ala16Val-SOD2 (dbSNP: rs4880) and Pro198Leu-GPx1 (dbSNP: rs1050450) polymorphisms were studied by allelic discrimination using fluorogenic probes and the 5'nuclease (TaqMan) assay. Twenty-four patients (with ICM (n = 8) or DCM (n = 16), age = 57.5 ± 10.7 years, LVEF = 25.3 ± 8.5%, NT-proBNP levels = 8540 ± 1703 ng/L) were included during a 15 month follow-up. OS parameters were significantly higher in patients than in controls. Distribution of MnSOD genotypes was 47% Val/Val-variant, 29.5% Ala/Val and 23.5% Ala/Ala-variants. Severity of CS was more important in patients with Val/Val-variant and can be put in parallel with NT-proBNP levels (Val/Val-variant: 11 310 ± 3875 ng/L vs Ala/Ala-variant: 6486 ± 1375 ng/L and Ala/Val-variant: 6004 ± 2228 ng/L; p < 0.05) and hemodynamic support duration (144.6 vs Ala/Val-variant: 108.8 h and Ala/Ala-variant: 52.5 h; p < 0.05) with a positive correlation (Spearman rho = 0.72, p < 0.05). Moreover, Val/Val-variant significantly influenced the mortality (Spearman rho = 0.67, p < 0.05), but not the morbidity (p = 0.3). Distribution of GPx genotypes was 64% Pro/Pro, 18% Pro/Leu and 18% Leu/Leu. GPx-variants influenced neither GPx activities nor cardiac events. In conclusion, CS was associated with markers of increased OS. GPx polymorphism did not influence the GPx activity. Only the Val-encoding MnSOD allele was significantly correlated with the severity and prognosis of CS.

Human kidney prolidase--purification, preincubation properties and immunological reactivity.

1. Prolidase I (EC 3.4.13.9) was purified from human kidney to SDS-PAGE homogeneity. The molecular weights of native and denatured purified enzyme were estimated to be 115,000 and 55,000, respectively. 2. Agarose electrophoresis revealed migration in the alpha 1 globulin region, and an isoelectric point (pl) of 4.65 was estimated by both isoelectric focusing (IEF) and the titration curve method. 3. Activation by preincubation for 24 hr at 37 degrees C with 1 mM MnCl2 was maintained throughout the purification steps, using gly-pro and phe-pro dipeptides as substrates. 4. Activation in the presence of gly-pro was higher (4.5- to 11-fold) than in the presence of phe-pro (1.3- to 2.3-fold). 5. Lineweaver-Burk plot consisted of one and two lines with gly-pro and phe-pro, respectively. Km, Vmax and the Vmax/Km ratio were increased and the two lines with phe-pro were conserved after prolonged preincubation. 6. A specific polyclonal antibody was raised in rabbits against the purified enzyme and immunoreactivity was investigated between rabbit antiserum and both prolidase I from various tissues and human kidney prolidase II. 7. Prolidase I from liver, erythrocytes and plasma was immunochemically identical to renal prolidase I. The polyclonal antibody did not react with prolidase II. 8. These results indicate that a specific immunoassay might be developed to investigate prolidase I protein in plasma and tissues from patients with prolidase deficiency and hepatic fibrosis.

Only prolidase I activity is present in human plasma.

1. After ion exchange chromatographic separation, liver prolidase exhibits two isoforms (prolidase I and II). 2. The activity of both was explored in human and rat tissues, and in normal and cytolytic human plasma. 3. The activity of prolidase I, eluted at the lowest ionic strength, was stimulated by 24 hr of preincubation with 1 mM MnCl2, but prolidase II activity was strongly inhibited by this long preincubation. In both normal and cytolytic human plasma, chromatographic separation also disclosed that only prolidase I activity was present. 4. This isoform displayed properties resembling those of liver and kidney prolidase I. 5. To explain the absence of prolidase II activity from the plasma, we tested the possibility that its tissue distribution differed. 6. However, this was not substantiated by the distribution found, or by the location, molecular weight and behavior of human liver prolidase II after neuraminidase treatment. 7. We also explored the hypothesis that plasma proteins inhibit prolidase II activity, and found that albumin almost abolished this activity after 6 hr incubation.

Standard treatment of alpha-tocopherol in Alagille patients with severe cholestasis is insufficient.

Alpha-tocopherol (alpha-T) is the most effective lipid-soluble antioxidant present in cells. We investigated the efficacy of alpha-T supplements for preventing lipid peroxidation in patients with Alagille syndrome, according to the severity of cholestasis. Patients were assigned to two groups on the basis of plasma bilirubin concentration (group I, bilirubin <100 microM; group II, bilirubin >100 microM). alpha-T concentrations were determined in plasma, in isolated lipoproteins, and in red blood cell membranes. In both groups of patients, alpha-T concentrations in plasma were similar to those in control subjects, but the distribution of alpha-T in lipoproteins was affected by the abnormal lipoprotein pattern in these patients. The efficacy of alpha-T was estimated by determining the amount of hydroperoxide produced from phosphatidylcholine and phosphatidylethanolamine (PE) molecular species owing to oxidative stress induced by lipoxygenase treatment. The concentrations of phosphatidylcholine molecular species and its corresponding hydroperoxides were significantly higher in both groups of patients. In group I, alpha-T and PE molecular species concentrations were similar to those in control subjects, but PE hydroperoxide concentrations were higher than those in the control subjects. In group II, alpha-T concentration was significantly lower and the concentrations of some PE molecular species and all PE hydroperoxides were lower than those in the control subjects. In conclusion, erythrocyte membrane alpha-T concentration was significantly lower only in patients with severe jaundice, despite alpha-T supplementation, raising the question as to whether the usual treatment was appropriate in this group.