Apoprotein Content Research Articles

THE CORRELATION PARAMETERS OF CARDIAC DYSFUNCTION ON THE BACKGROUND OF STEATOHEPATOSIS

Introduction. Coronary heart disease (CHD) and its complications in the modern world are one of the main causes of disability and mortality of people of different ages [1-3]. Fatty liver disease - steatohepatosis - is an important factor in the development and progression of CAD and increases the risk of developing type 2 diabetes. On the other hand, information on cardiac dysfunction in patients with coronary heart disease with manifestations of steatohepatosis is insufficient and contradictory, the diagnosis, clinical features and treatment of this pathology are not accurate enough.
 The aim of the study. Determination of the correlations of the studied indicators in patients with coronary artery disease complicated by fatty liver disease.
 Research methods. Fatty infiltration of the liver was diagnosed using ultrasound of the abdominal cavity (EUB-6000 scanner; Hitachi Medical Corporation, Japan). The content of NT-proBNP was determined on StatFax-303, the activity of enzymes and indicators of carbohydrate and lipid metabolism were determined using the CRPLX "Roche Diagnostics" reagent set on the COBAS INTEGRA 400 plus automatic analyzer.
 In order to identify correlations, the coefficient of linear correlation |r| was determined between all studied indicators. The strength of the relationship was estimated by the absolute value of the Pearson linear correlation coefficient, considering that at r <0.25 the relationship is weak, 0.25 <r< 0.75 - a relationship of medium strength, r ˃ 0.75 - correlation the bond is strong. We used the STATISTICA 6.0 software package (StatSoft, USA) [8].
 Results and discussion. Strong probable positive correlations were found between glucose concentration and the content of triacylglycans, glycated hemoglobin, atherogenicity coefficient, and lipase activity.
 Strong probable positive correlations were found between the activity of pancreatic amylase and the concentration of creatinine, C-reactive protein, interleukin 1β, NT-proBNP, and ALT activity.
 Strong probable positive correlations were found between the concentration of NT-proBNP and the content of uric acid, glucose, C-reactive protein, apoprotein A, interleukin 10, LDH activity, and alkaline phosphatase.
 Strong probable positive correlations were found between the concentration of creatinine and the content of total cholesterol, the content of HDL-cholesterol, LDL-cholesterol, and lipase activity.
 Strong probable positive correlations were found between the concentration of total cholesterol and the content of HDL-cholesterol, creatinine, and lipase activit
 Conclusions. Steatohepatosis is a factor in the development and progression of coronary heart disease and probably increases the risk of developing type 2 diabetes. In the group of patients with steatohepatosis, reliable strong correlation links of carbohydrate metabolism with other parameters were revealed. In the group of patients with on the background of steatohepatosis, significant strong correlation of the level of NT-proBNP, cholesterol and creatinine with other parameters was found.

Epicardial fat thickness and biomarkers of inflammation in patients with stable coronary artery disease: correlation with the severity of coronary atherosclerosis

Aim. To study the relationship between the epicardial fat thickness (EFT), biomarkers of inflammation and metabolic dysfunction in patients with coronary artery disease (CAD) and various severity of coronary atherosclerosis.Material and methods. The study consisted of 89 patients (47 men and 42 women) with stable CAD at the age of 62,2±6,5 years, who assessed the presence and severity of coronary atherosclerosis according to angiography with the calculation of the Gensini Score (GS). We conducted an ultrasonic evaluation of the EFT. We determined the content of glucose, lipid fractions, apoproteins, pro-inflammatory cytokines and adipokines, C-reactive protein by a highly sensitive method (hsCRP).Results. In the total sample of patients, the median GS index was 13,5 (3,5; 43) points, the median EFT was 4,93 (3,95; 6,0) mm, the median hsCRP was 2,1 (1,02; 3,65) mg/l. There were no correlation relationships between the GS index and the body mass index, waist circumference, EFT, hsCRP, lipid and carbohydrate metabolism in the general group of patients. In the course of linear regression analysis, an independent contribution of hsCRP >2,1 mg/l to the formation of the first two tertiles of a sample of GS values was established (0≤ GS ≤28, paired linear regression hsCRP at GS β=0,55, p=0,0221), whereas in patients with GS values from the third tertile (GS >28 points), the growth of this parameter had an independent association with an increase in the EFT (estimation of the coefficient of paired linear regression of the EFT on GS β=0,56, p=0,0015). The range of hsCRP changes did not affect the value of the β coefficient in paired linear regression models in patients with GS >28 points (n=29) and in the subgroup of patients with GS >28 and hsCRP >2,1 mg/l (n=18).Conclusion. The absence of an independent association between EFT and minor or moderate severity of atherosclerotic lesions of the coronary arteries (for GS ≤28 points) in patients with CAD, while an independent marker of GS index increase is higher than 2,1 mg/l. An independent contribution to the formation of a severe coronary atherosclerosis (with a GS value of >28 points) is equally made by thickening of EFT, and a moderately elevated level of hsCRP.

LpA-II:B:C:D:E: a new immunochemically-defined acute phase lipoprotein in humans

BackgroundPrevious studies of lipoproteins in patients with sepsis have been performed on density fractions isolated by conventional ultracentrifugation that are heterogeneous and provide no information about the cargo of apoproteins present in the immunochemically distinct subclasses that populate the density classes. Since apoproteins are now known to have important roles in host defense, we have separated these subclasses according to their apoprotein content and characterized their changes during experimental endotoxemia in human volunteers.MethodsWe have studied apoB- and apoA containing lipoprotein subclasses in twelve healthy male volunteers before and for 8 h after a single dose of endotoxin (ET; 2 μg/kg) to stimulate inflammation.ResultsAfter endotoxin, TG, TC, apoB and the apoB-containing lipoprotein cholesterol-rich subclass LpB and two of the three triglyceride-rich subclasses (TGRLP: Lp:B:C, LpB:C:E+ LpB:E) all declined. In contrast, the third TGRLP, LpA-II:B:C:D:E (“complex particle”), after reaching a nadir at 4 h rose 49% above baseline, p = .006 at 8 h and became the dominant particle in the TGRLP pool. This increment exceeds the threshold of > 25% change required for designation as an acute phase protein. Simultaneous decreases in LpA-I:A-II and LpB:C:E + LpB:E suggest that these subclasses undergo post-translational modification and contribute to the formation of new LpA-II:B:C:D:E particles.ConclusionsWe have identified a new acute phase lipoprotein whose apoprotein constituents have metabolic and immunoregulatory properties applicable to host defense that make it well constituted to engage in the APR.

Effects of Torcetrapib and Statin Treatment on ApoC-III and Apoprotein-Defined Lipoprotein Subclasses (from the ILLUMINATE Trial)

In the ILLUMINATE Trial, treatment with the cholesteryl ester transfer protein inhibitor torcetrapib resulted in a significant increase in both atherosclerotic cardiovascular disease events and total mortality which was not explained by changes in the routinely measured plasma lipids. To determine whether alterations in lipoproteins defined by their apoprotein content that are not estimated with conventional laboratory methods contributed to these unexpected events, we measured the apoB- and apoA-containing subclasses in a subgroup of ILLUMINATE participants. We find that torcetrapib treatment significantly increasedthe high-density lipoprotein subclasses LpA-I and LpA-I:A-II equally (p <0.0001) and the apoC-III content of high-density lipoprotein (p <0.001) without altering theapoB-containing subclasses. In conclusion, these findings provide further evidence thatthe untoward effects of torcetrapib were attributable to off-target effects and not related to disturbances in lipoprotein transport.

Apolipoprotein B levels and its relation to lipid profile in type II - Diabetes Mellitus; Comparative study

Background Type 2 diabetes mellitus is not only associated with carbohydrate metabolic disturbances but also been associated with dyslipidemia comprising of multiple lipoprotein disorders which increases the risk of cardiovascular morbidity and mortality. Apolipoprotein A-1 Apo A1 and apolipoprotein B Apo B are the main structural proteins of the circulating HDL particles and atherogenic lipoproteins respectively. Since apo B is the major apoprotein content of the atherogenic lipoprotein study of apo B levels may help in identifying diabetics who are at risk of developing cardiovascular morbidity even before there is alteration in the levels of LDL and HDL cholesterol.Objectives 1 To estimate serum total cholesterol triglycerides HDL cholesterol LDL cholesterol and Apo B levels in type 2 diabetes mellitus and comparing them with controls 2 To correlate the Apo B levels with total cholesterol triglycerides HDL cholesterol and LDL cholesterol in cases of type 2 diabetes and controls.Methods Subjects were from the patients attending Medical OPD amp Diabetology OPD of R.L.Jalappa Hospital and Research Centre Kolar. The subjects of either sex within the age group of 40 to 65 years were included for the study its Comparative study Sample size of 45 and divisions of following groups were made Group A - Proven cases of Type 2 diabetes mellitus under poor control Group B - Diabetic Controls Type 2 diabetes mellitus patients under control based on glycated hemoglobin levels amp Group C - Normal Healthy Controls.Results The levels of apo B is found to be increased in uncontrolled diabetes patients compared to the controlled diabetics and healthy controls. Correlation of Apo B showed a positive significant correlation with total cholesterol and LDL cholesterol in all the groups.Conclusion Apolipoprotein estimations give more insight to on-going metabolic changes occurring with circulating lipids. Lipid alterations are reflected better by Apo B estimation in type 2 diabetes mellitus and predicts the risk of associated complications with it.

BAL の標準的洗浄法に関する検討 : CT 像からみた洗浄領域とフラクション分析

BAL の標準的洗浄法に関する検討 : CT 像からみた洗浄領域とフラクション分析

Photochemical activity and the structure of chloroplasts in Arabidopsis thaliana L. mutants deficient in phytochrome A and B

The reduced content of photoreceptors, such as phytochromes, can decrease the efficiency of photosynthesis and activity of the photosystem II (PSII). For the confirmation of this hypothesis, the effect of deficiency in both phytochromes (Phy) A and B (double mutant, DM) in 7-27-day-old Arabidopsis thaliana plants on the photosynthetic activity was studied in absence and presence of UV-A radiation as a stress factor. The DM with reduced content of apoproteins of PhyA and PhyB and wild type (WT) plants with were grown in white and red light (WL and RL, respectively) of high (130μmol quanta m-2 s-1) and low (40μmol quanta m-2 s-1) intensity. For DM and WT grown in WL, no notable difference in the photochemical activity of PSII was observed. However, the resistance of the photosynthetic apparatus (PA) to UV-A and the rate of photosynthesis under light saturation were lower in the DM compared to those in the WT. Growth in RL, when the photoreceptors of blue light-cryptochromes-are inactive, resulted in the significant decrease of the photochemical activity of PSII in DM compared to that in WT including amounts of QB-non-reducing complexes of PSII and noticeable enhancement of thermal dissipation of absorbed light energy. In addition, marked distortion of the thylakoid membrane structure was observed for DM grown in RL. It is suggested that not only PhyA and PhyB but also cryptochromes are necessary for normal functioning of the PA and formation of the mechanisms of its resistance to UV-radiation.

Apolipoprotein A5 ‐ enriched HDL is Poor Acceptor for Cholesterol in in vitro Efflux Study

High density lipoprotein (HDL) plays central role in reverse cholesterol (CH) transport (RCT). Although apolipoprotein A5, a modulator of triglyceride‐rich lipoprotein metabolism, is abundant on HDL its effect on HDL‐driven RCT has not been elucidated. To test this macrophages loaded with 3H‐ CH were incubated with whole plasma, total HDL (d=1.063–1.21), HDL3 (d=1.063–1.09), preβ‐HDL (d=1.15–1.21) from apoA5 transgenic (A5tg) and from background mice. Lipoproteins from A5tg mice were associated with significantly (p&lt;0.05) lower CH efflux (expressed as % of medium/medium + cells cpm): 54.0 ± 4.0 vs. 35 ± 2.0 in the presence of whole plasma, 19.9 ± 4.0 vs. 12.2 ± 2.0 in the presence of total HDL, 28 ± 5.0 vs. 16.0 ± 3.0 in the presence of HDL3, 8.2 ± 2.0 vs. 4.2 ± 1.0 in the presence of preβ‐HDL. When we use HDL2 (d=1.09–1.15) as acceptor, CH efflux for A5tg mice was comparable to background FVB mice. Additionally, no difference in CH efflux between the two mice strains was demonstrated in experiments that used as acceptor very low density lipoprotein (VLDL) ‐ a lipoprotein that does not involve lecithin‐cholesterol acyltransferase (LCAT) activity. While potential underlying case include differences between the groups in both lipid and apoprotein content of HDL, the lack of difference between the groups in the VLDL experiments supports the possibility that interference of apoA5 with the activity of LCAT is a likely mechanism.

Disruption of the Gene forCYP1A2, which Is Expressed Primarily in Liver, Leads to Differential Regulation of Hepatic and Pulmonary Mouse CYP1A1 Expression and Augmented Human CYP1A1 Transcriptional Activation in Response to 3-Methylcholanthrene In Vivo

The cytochrome P4501A (CYP1A) enzymes play important roles in the metabolic activation and detoxification of numerous environmental carcinogens, including polycyclic aromatic hydrocarbons (PAHs). In this study, we tested the hypothesis that hepatic CYP1A2 differentially regulates mouse hepatic and pulmonary CYP1A1 expression and suppresses transcriptional activation of human CYP1A1 (hCYP1A1) promoter in response to 3-methylcholanthrene (MC) in vivo. Administration of wild-type (WT) (C57BL/6J) or Cyp1a2-null mice with a single dose of MC (100 μmol/kg i.p.) caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein content, and mRNA levels 1 day after carcinogen withdrawal compared with vehicle-treated controls. The induction persisted in the WT, but not Cyp1a2-null, animals, for up to 15 days. In the lung, MC caused persistent CYP1A1 induction for up to 8 days in both genotypes, with Cyp1a2-null mice displaying a greater extent of CYP1A1 expression. It is noteworthy that MC caused significant augmentation of human CYP1A1 promoter activation in transgenic mice expressing the hCYP1A1 and the reporter luciferase gene on a Cyp1a2-null background, compared with transgenic mice on the WT background. In contrast, the mouse endogenous hepatic, but not pulmonary, persistent CYP1A1 expression was repressed by MC in the hCYP1A1-Cyp1a2-null mice. Liquid chromatography-mass spectrometry experiments showed that CYP1A2 catalyzed the formation of 1-hydroxy-3-MC and/or 2-hydroxy-3-MC, a metabolite that may contribute to the regulation of CYP1A1 expression. In conclusion, the results suggest that CYP1A2 plays a pivotal role in the regulation of hepatic and pulmonary CYP1A1 by PAHs, a phenomenon that potentially has important implications for PAH-mediated carcinogenesis.

Lung surfactant apoprotein (LSP) content of sputum samples from patients with chronic airway diseases

LSP contents of sputum samples from patients with chronic airway diseases were measured by an enzyme-linked immunosorbent assay (ELISA) kit which was designed by Kuroki et al to examine whether a substance identical to lung surfactant contained in alveolar lining layer, is also contained in respiratory tract fluid or not in the ELISA kit. One antibody to LSP was conjugated to peroxidase and another one to LSP was fixed onto a bead. A neo-anionic detergent, Triton X-100 and an anionic detergent, sodium dodecyl sulfate (SDS) were added to extraction medium to separate LSP from lung surfactant, and LSP reaction of sputum sample was maximal when the ratio of Triton X-100 to SDS was in range of 1 to 4. Airway mucous glycoprotein (AMG) purified from sputum sample did not show any LSP reaction. In CsCl density gradient ultracentrifugation of whole sputum, the LSP reaction was detectable only in the top fraction with density of about 1.40 and AMG was located in the fraction with a density of about 1.50. These results indicate that the LSP reaction of sputum sample is not due to false reaction caused by nonspecific binding of viscous AMG to the two antibodies to LSP, but to the existence of LSP. Therefore it was concluded that lung surfactant is contained in respiratory tract fluid. In general, the LSP concentrations in sputum samples were lower in purulent sputa than in mucoid or mucopurulent sputa, and lower in patients with diffuse panbronchiolitis and bronchiectasia than in those with pulmonary emphysema and chronic bronchitis. It was shown that LSP was hydrolyzed by neutrophil leucocyte homogenate. These results suggest that LSP content of sputum is influenced by various factors such as infection and disease in the respiratory tract, and thus is useful to estimate pathological states of chronic airway diseases.

Persistent induction of cytochrome P4501A1 in human hepatoma cells by 3-methylcholanthrene: evidence for sustained transcriptional activation of the CYP1A1 promoter.

Cytochrome P450 (P450)1A1 plays a critical role in the metabolic activation and detoxification of polycyclic aromatic hydrocarbons (PAHs), many of which are potent human carcinogens. In this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1A1 expression in human hepatoma cells (HepG2) and that this phenomenon is mediated by sustained transcriptional activation of the CYP1A1 promoter. Treatment of HepG2 cells with MC resulted in marked induction (8-20-fold) of ethoxyresorufin O-de-ethylase activities, CYP1A1 apoprotein contents, and mRNA levels, which persisted for up to 96 h. MC also caused sustained transcriptional activation of the human CYP1A1 promoter for up to 96 h, as inferred from transient transfection experiments. Experiments with deletion constructs indicated that Ah response elements located at -886, -974, and -1047, but not -491, nucleotides from the start site, contributed to the sustained transcriptional activation of the CYP1A1 promoter. Electrophoretic mobility-shift and chromatin immunoprecipitation assays suggested that prolonged CYP1A1 induction was mediated by Ah receptor (AHR)-independent mechanisms. Experiments with [3H]MC and liquid chromatography-tandem mass spectrometry demonstrated rapid elimination of MC and its metabolites from the cells by 12 to 24 h, suggesting that these compounds did not elicit sustained CYP1A1 induction via the classical AHR-mediated pathway. In conclusion, the results of this study support the hypothesis that MC causes persistent induction of CYP1A1 in human hepatoma cells by mechanisms entailing sustained transcriptional activation of the CYP1A1 promoter via AHR-independent mechanisms. These observations have important implications for human carcinogenesis mediated by PAHs.

Myocardial metabolism of triacylglycerol‐rich lipoproteins in type 2 diabetes

Cardiac utilisation of very-low-density lipoprotein (VLDL) and chylomicrons (CM) was investigated in the ZDF rat model of type 2 diabetes, in order to define the role of triacylglycerol (TAG) metabolism in the development of contractile dysfunction. Hearts from obese diabetic and lean littermate control rats were perfused with VLDL and CM from diabetic and control rats. Metabolic fate of the lipoprotein TAG and contractile function were examined. Myocardial utilisation of both VLDL- and CM-TAG was increased in the diabetic state. Diabetic hearts oxidised diabetic lipoprotein-TAG to a greater extent than control lipoproteins; glucose oxidation was decreased. There was no difference in lipoprotein-TAG assimilation into diabetic heart lipids; diabetic lipoproteins were, however, a poor substrate for control heart tissue lipid accumulation. Although the proportion of exogenous lipid incorporated into tissue TAG was increased in diabetic hearts perfused with control lipoproteins, this effect was not seen in diabetic hearts perfused with diabetic lipoproteins. Myocardial heparin-releasable lipoprotein lipase (LPL) activity was moderately increased in the diabetic state, and diabetic lipoproteins increased tissue-residual LPL activity. Cardiac hydraulic work was decreased only in diabetic hearts perfused with diabetic CM. Compositional analysis of diabetic variant lipoproteins indicated changes in size and apoprotein content. Alterations in cardiac TAG-rich lipoprotein metabolism in type 2 diabetes are due to changes in both the diabetic myocardium and the diabetic lipoprotein particle; decreased contractile function is not related to cardiac lipid accumulation from TAG-rich lipoproteins but may be associated with changes in TAG-fatty acid oxidation.

Transcriptional responses of Mycobacterium tuberculosis to lung surfactant

This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mixture of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30 min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2 h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (≤20 genes were induced) suggesting that interactions among multiple lipid–protein components of WLS may contribute to its effects on Mtb transcription.

Quantitation of Human Cytochrome P450 2D6 Protein with Immunoblot and Mass Spectrometry Analysis

Accurate quantification of cytochrome P450 (P450) protein contents is essential for reliable assessment of drug safety, including the prediction of in vivo clearance from in vitro metabolism data, which may be hampered by the use of uncharacterized standards and existence of unknown allelic isozymes. Therefore, this study aimed to delineate the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compare immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification of CYP2D6.1 and CYP2D6.2 allelic isozymes. Holoprotein content of in-house purified CYP2D6 isozymes was determined according to carbon monoxide difference spectrum, and total protein was quantified with bicinchoninic acid protein assay. Holoprotein/total CYP2D6 protein ratio was markedly higher for purified CYP2D6.1 (71.0%) than that calculated for CYP2D6.1 Supersomes (35.5%), resulting in distinct linear calibration range (0.05-0.50 versus 0.025-0.25 pmol) that was determined by densitometric analysis of immunoblot bands. Likewise, purified CYP2D6.2 and CYP2D6.10 and the CYP2D6.10 Supersomes all showed different holoprotein/total CYP2D6 protein ratios and distinct immunoblot linear calibration ranges. In contrast to immunoblot, nano-LC/MS readily distinguished CYP2D6.2 (R296C and S486T) from CYP2D6.1 by isoform-specific proteolytic peptides that contain the altered amino acid residues. In addition, relative quantitation of the two allelic isozymes was successfully achieved with label-free protein quantification, consistent with the nominated ratio. Because immunoblot and nano-LC/MS analyses measure total P450 protein (holoprotein and apoprotein) in a sample, complete understanding of holoprotein and apoprotein contents in P450 standards is desired toward reliable quantification. Our data also suggest that nano-LC/MS not only facilitates P450 quantitation but also provides genotypic information.

Regulation of cytochrome P4501A1 expression by hyperoxia in human lung cell lines: Implications for hyperoxic lung injury

Supplemental oxygen, used to treat pulmonary insufficiency in newborns, contributes to the development of bronchopulmonary dysplasia (BPD). Cytochrome P4501A enzymes are induced by hyperoxia in animal models, but their role in human systems is unknown. Here we investigated the molecular mechanisms of induction of CYP1A1 by hyperoxia in human lung cell lines. Three human lung cell lines were exposed to hyperoxia (95% O2) for 0–72 h, and CYP1A1 activities, apoprotein contents, and mRNA levels were determined. Hyperoxia significantly induced CYP1A1 activity and protein contents (2–4 fold), and mRNA levels (30–40 fold) over control in each cell line. Transfection of a CYP1A1 promoter/luciferase reporter construct, followed by hyperoxia (4–72 h), showed marked (2–6 fold) induction of luciferase expression. EMSA and siRNA experiments strongly suggest that the Ah receptor (AHR) is involved in the hyperoxic induction of CYP1A1. MTT reduction assays showed attenuation of cell injury with the CYP1A1 inducer beta-naphthoflavone (BNF). Our results strongly suggest that hyperoxia transcriptionally activates CYP1A1 expression in human lung cell lines by AHR-dependent mechanisms, and that CYP1A1 induction is associated with decreased toxicity. This novel finding of induction of CYP1A1 in the absence of exogenous AHR ligands could lead to novel interventions in the treatment of BPD.

CYP4A11 is repressed by retinoic acid in human liver cells

CYP4A11, the major fatty acid ω-hydroxylase in human liver is involved in the balance of lipids, but its role and regulation are both poorly understood. We studied the effects of retinoids on the regulation of CYP4A11 in the human hepatoma cell line HepaRG. Treatment of HepaRG cells with all- trans-retinoic acid resulted in a strong decrease in CYP4A11 gene expression and apoprotein content and, furthermore, was associated with a 50% decrease in the microsomal lauric acid hydroxylation activity. Such a strong suppression of CYP4A11 expression by retinoids could have a major impact on fatty acid metabolism in the liver.

Intraperitoneal injection ofd-galactosamine provides a potent cell proliferation stimulus for the detection of initiation activities of chemicals in rat liver

In an in vivo 5-week initiation assay model, chemical hepatectomy by hepato-toxicant administration was utilized as a cell proliferation stimulus as an alternative to the two-thirds partial hepatectomy. The study investigated the effect of an intraperitoneal (i.p.) injection of D-galactosamine (D-gal) for this purpose in a medium-term liver bioassay, with a further focus on cell proliferation kinetics and cytochrome P450 (CYP) expression. In experiment I, cell proliferation in rat liver after a single administration of D-gal (700 mg kg(-1), i.p.) was analysed by the bromodeoxyuridine (BrdU) labeling method, and CYP isozymes were quantified by immunoblotting. In experiment II, the induction of glutathione S-transferase placental form (GST-P) positive foci by 1,2-dimethylhydrazine (DMH) was evaluated in a modified in vivo 5-week initiation assay model. At 84 hours after single administration of d-gal (i.p.) the BrdU index was markedly elevated (27.5% +/- 9.5%). Although CYP 2E1 and 1A2 apoprotein contents decreased transiently to less than 20% of the control level, subsequently they recovered to 60% and 40% of the control level, respectively, at 84 hours. Induction of GST-P positive foci in the group given DMH at 84 hours after a single administration of d-gal was significantly greater than in the control group, correlating with the kinetics of cell proliferation. In conclusion, the sensitivity of the present initiation assay using D-gal i.p. is high, so that D-gal i.p. can be considered an effective cell proliferation stimulus.

A rapid methodology for the isolation of intermediate-density lipoprotein: characterization of lipid composition and apoprotein content

Background Intermediate-density lipoprotein (IDL) is a structurally related precursor of low-density lipoprotein (LDL). Although found in significantly lower levels, extensive evidence suggests that IDL shares LDL's capacity to promote atherosclerosis. To assist further investigation into IDL's composition and physiological relevance, we have established a rapid method to extract IDL from plasma. Methods IDL was isolated from plasma by sequential floatation ultracentrifugation in 3 h, a significantly shorter isolation time than previously published methods. Apoproteins (apo) B 100, CIII, and E, together with the level of albumin contamination, were quantified using single radial immunodiffusion. The lipid composition of IDL was measured using automated enzyme assays. Results The percent recovery of lipid from all lipoprotein fractions (VLDL+IDL+LDL+HDL) was 97.0±4.9% when compared to total plasma lipid. IDL had a reduced concentration of apo CIII, apo E, triglyceride, and free cholesterol, and had a higher concentration of apo B 100, cholesterol ester, and phospholipid when compared to VLDL. Pure IDL migrated in advance of LDL during agarose electrophoresis. Conclusions This rapid technique minimizes damage to the integrity of IDL and yields sufficient quantities to allow accurate assessment of composition and susceptibility to in vitro oxidation, and thus facilitates further investigation of IDL in the development of atherosclerosis.

Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation.

The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes. A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles. CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting. 4-Hydroxy-CPA and hydroxy-bupropion were quantified by using HPLC and LC-MS, respectively. 7-Ethoxy-4-trifluoromethyl coumarin O-deethylase activity was measured fluorometrically. The frequencies of CYP2B6*5 and CYP2B6*6 mutant alleles were 9.0 and 16.4%, respectively. CYP2B6 protein expression was detected in 80% of the samples, with a large variation (0.003-2.234, arbitrary units). There was a high correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation (n=55, r=0.81, P<0.0001). When based on the CYP2B6 apoprotein levels, the *6 carriers had significantly higher CPA 4-hydroxylation (P<0.05). CPA 4-hydroxylation also correlated significantly with other CYP2B6-specific reactions (n=20, P<0.0001). V(max) and K(m) for CPA 4-hydroxylation in recombinant CYP2B6 enzyme were 338 nmol/min/nmol enzyme and 1.4 mM, respectively. CYP2B6 showed much higher in vitro intrinsic clearance than previously observed in recombinant CYP2C19 and CYP2C9 variants in yeast expression system. Our results demonstrate that the polymorphic CYP2B6 is a major enzyme in the bioactivation of CPA. Moreover, we identified a strong impact of CYP2B6*6 on CPA 4-hydroxylation.

Trifluoroacetylated adducts in spermatozoa, testes, liver and plasma and CYP2E1 induction in rats after subchronic inhalatory exposure to halothane

The induction of cytochrome P450 (CYP) 2E1 in testes and liver and the presence of trifluoroacetylated (TFA) adducts in spermatozoa, testes, liver and plasma were investigated in rats subchronically exposed by inhalation to halothane (15 ppm/4 h/day/5 days/week/9 weeks). After halothane exposure, p-nitrophenol hydroxylase ( p-NPH) activity increased 3.2-fold and CYP2E1 apo-protein content 7-fold in testes, whereas in liver, p-NPH increased 2.3-fold and CYP2E1 apoprotein content 1.4-fold. These results suggest a differential inductive effect of halothane on CYP2E1 in these tissues. Moreover, TFA adducts were present in microsomes of testis and liver and in plasma of halothane-treated rats. The immunoblot analysis of testicular microsomes showed two intense TFA protein bands of 63 and 59 kDa, whereas in liver three intense bands of 100, 76 and 63 kDa were observed. Bands of similar molecular weights to those observed in liver were detected in the plasma of halothane-treated animals. In addition, TFA adducts were detected by immunofluorescence in spermatozoa, probably in the acrosome and/or perinuclear theca region, and in the distal tail of spermatozoa. The increase in CYP2E1 apoprotein and p-NPH activity observed in testis and liver microsomes suggests that halothane induces its own biotransformation both hepatically and extrahepatically and in addition, that the nature of the TFA adducts will depend on the proteins present in each tissue. Also, the presence of TFA adducts in spermatozoa may result from the activation of halothane in the reproductive tract. The detailed mechanism of TFA adduct formation and its consequences on the spermatozoa function remain to be fully clarified.