Evaluation of cholesterol absorption in rats using markers labeled with stable isotopes. Effect of complete bile diversion

BACKGROUND/AIMS: An easily performed method to measure cholesterol absorption with isotope labeled cholesterol and beta-sitostanol in humans is described. The first aim of the study was to show whether this method can also be used in rats. Secondly, to see whether complete bile diversion results in a complete loss of cholesterol absorption. METHODOLOGY: Cholesterol absorption was evaluated in rats by the constant isotope feeding method using [2H6]cholesterol and [2H4]sitostanol as markers. Fecal samples were analyzed by gas-chromatography/mass spectrometry. RESULTS: In 8 rats with intact enterohepatic circulation of bile acids, cholesterol absorption averaged 61 (3% (SD) (range: 54-69%)). Complete bile diversion was followed by an almost total loss of cholesterol absorption (5.5+/-0.6%, range: 2.4-6.9%, n=7). CONCLUSIONS: The results indicate that deuterated cholesterol and deuterated sitostanol are reliable markers for measurement of cholesterol absorption in rats and that bile acids are essential for cholesterol absorption.     

Sequences within the amino terminus of ApoB100 mediate its noncovalent association with apo(a)

Although sequences within the C terminus of apolipoprotein B (apoB) have been implicated in the formation of covalent lipoprotein(a) [Lp(a)] particles, sequences in apoB that mediate initial noncovalent interaction with apo(a) remain to be characterized. To address this question, we have used an affinity chromatography method in which 2 recombinant forms of apo(a) [r-apo(a); either a 17-kringle form (17K) or a derivative containing apo(a) kringle IV types 5-8] have been immobilized onto Sepharose beads. Conditioned media from rat hepatoma (McA-RH7777) cell lines stably expressing various carboxyl-terminally truncated forms of human apoB (ranging from full-length apoB to apoB15) were applied to the r-apo(a) affinity columns; the columns were subsequently washed and eluted with epsilon-aminocaproic acid (epsilon-ACA). Specific binding was quantified by Western blot analysis of column fractions. Of the apoB truncations examined, apoB94, apoB42, apoB37, and apoB29 exhibited complete specific binding to 17K r-apo(a). Only approximately 50% binding was observed for apoB18, whereas essentially no detectable binding was observed with apoB15. In all cases, similar results were obtained when the r-apo(a) kringle IV types 5-8-Sepharose column was used. Additionally, substitution of proline for epsilon-ACA as the eluent resulted in similar column profiles with either r-apo(a) affinity column. We also demonstrated that apoB48 present in chylomicrons bound completely to the 17K column in an epsilon-ACA-dependent manner. Taken together, these results represent the first demonstration that N-terminal sequences in apoB between amino acid residues 680 (apoB15) and 781 (apoB18) are essential for noncovalent association with apo(a) and that these sequences interact with domain(s) present within apo(a) kringle IV types 5-8.     

Plasma amino acids and nitrogen retention of human subjects who consumed isonitrogenous diets containing rice and wheat or their constituent amino acids with and without additional lysine

Six diets furnished 6.0 g of nitrogen to adult human subjects as follows: a combination of rice and wheat; mixtures in which either 100 or 50% of the amino acids in rice and wheat were replaced by their constituent amino acids; and similar diets in which lysine was increased from 1.2 to 1.8 g. Nitrogen retention was greater (P less than 0.01) and concentrations of several amino acids in plasma were lower when the cereals were supplemented with lysine than when all other diets were fed. Data obtained with mixtures of amino acids apparently cannot always be used interchangeably with that from cereal-based diets, even when amino acid content is the same. Changes in plasma amino acids are a useful indicator of utilization of dietary amono acids when nitrogen balance also is determined.     

Protection of B lymphocyte hybridoma against starvation-induced apoptosis: survival-signal role of some amino acids

Immunofluorescence microscopic observations indicated that a monoclonal antibody, Vmp 18, raised against the peptide 199-208 of murine interleukin 1 alpha, cross-reacted with an antigenic determinant of Drosophila thorax muscles. Immunoelectron microscopic analysis showed that the gold particles were mainly localized in the Z-line which is the attachment site of thin filaments from adjacent sarcomeres. On the contrary, the antibody failed to mark the Z-line in vertebrate skeletal muscle. A Western blot of total protein extract from Drosophila thorax muscles bound a protein of 43 kDa. Our observations suggest that the Vmp 18 antibody could contribute to clarify the composition of the Z-line in insect's flight muscles.     

The pattern of apolipoprotein B100 containing lipoprotein subclasses produced by the isolated visceral rat yolk sac depends on developmental stage and fatty acid availability

Explants of visceral rat yolk sacs from gestational days 16, 18 and 22 were used for studying developmental changes of secretion and density distribution of lipoproteins, particularly of those containing apoB. Moreover, the influence of fatty acid supply on the amount and density distribution of secreted apolipoproteins was studied on day 18 of gestation. Active lipoprotein production was observed in yolk sacs taken on days 16 and 18 of gestation. It declined considerably on day 22 of gestation in parallel with the production of total protein, triacylglycerols and cholesterol. On all gestational days, apoB floated mainly in the LDL range ( > or = 70%) with differences in the distribution pattern of LDL subclasses. The lowest density of secreted LDL was found on day 18 of gestation (peak at d = 1.025 g/ml) followed by day 16 (peak at d = 1.035 g/ml) and day 22 of gestation (peak at d = 1.045 g/ml). ApoAIV, apoE and apoAI floated exclusively in the HDL range with a peak at d = 1.089 g/ml independently of the gestational day. After incubation of yolk sacs from the 18th day of gestation with 0.4 mM or 0.8 mM oleate, the density of secreted apoB containing particles was decreased (peaks in the VLDL and IDL density range), whereas palmitate in the same concentrations caused a redistribution of secreted apoB toward higher densities (peaks at d > or = 1.032 g/ml). Taken together, the data provide evidence that the density of LDL subclasses produced by isolated yolk sacs between days 16 and 22 of gestation depended on the gestational stage. Moreover, addition of unsaturated or saturated fatty acids to the organ culture differently affected the secretory rate and the density of lipoproteins delivered by yolk sacs on day 18 of gestation.     

Determination of total and specific IgE using UNICAP 100: comparative study with the CAP system

The methods of serum determination of specific IgE to different allergens have showed lower diagnostic sensitivity than the alternative in vivo methods, the skin tests. The CAP system from Pharmacia, owing to its solid phase, has ameliorated this disadvantage, showing in various studies greater diagnostic sensitivity than the classic RAST, without affecting specificity. However, this system is still semi-automatic and requires daily calibration. UNICAP 100 is a completely automatic autoanalyser for total IgE, specific IgE and Eosinophil Cationic Protein, which combines the high sensitivity of the CAP system with complete automation and monthly calibration. The aim of the present study is to assess the practicality and reliability of UNICAP 100, when compared to the CAP System, for the determination of total IgE and specific IgE, as well as the sensitivity and diagnostic specificity; using as a reference skin tests in 150 paediatric patients. The coefficients of variation in the study of intraseries imprecision ranged between 2.1% and 3.6% for total IgE and between 2.2% and 5.1% for specific IgE, depending on the allergen and the level studied. The intraseries imprecision ranged between 3.3% and 7.7% for total IgE and between 5.2% and 8.9% for specific IgE. The coefficients of correlation of the study of interchangeability with the results of the CAP System varied between 0.985 and 0.998, all the allergens tested (9) being interchangeable. Finally, the diagnostic sensitivity varied between 70% and 95% and the specificity between 87% and 100%. In conclusion, UNICAP 100 showed results that were interchangeable with the CAP System, noticeably improving the benefits owing to its complete automation and its calibration system.     

An international study on sleep disorders in the general population: methodological aspects of the use of the Sleep-EVAL system

The comparability among epidemiological surveys of sleep disorders has been encumbered because of the array of methodologies used from study to study. The present international initiative addresses this limitation. Many such studies using the exact same methodology are being completed in six European countries (France, the United Kingdom, Germany, Italy, Portugal, and Spain), two Canadian cities (metropolitan areas of Montreal and Toronto), New York State, and the city of San Francisco. These surveys have been undertaken with the aim of documenting the prevalence of sleep disorders in the general population according to criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) and the International Classification of Sleep Disorders (ICSD-90). Data are gathered over the telephone by lay interviewers using the Sleep-EVAL expert system. This paper describes the methodology involved in the realization of these studies. Sample design and selection procedures are discussed.     

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.     

Dimensionality of depression in acute schizophrenia: a methodological study using the Bech-Rafaelsen Melancholia Scale (BRMES)

Ca2+ plays an essential role in pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated growth hormone (GH) secretion from porcine somatotropes. Here, Indo-1 microfluorimetry was used to investigate the dynamics of free cytosolic Ca2+ concentration ([Ca2+]i) in single porcine somatotropes in response to PACAP38 and PACAP27. We also evaluated the relative contributions of extra- and intracellular Ca2+ sources and of cAMP-dependent protein kinase (PKA) and phospholipase C (PLC). A high proportion of somatotropes responded to PACAP38 (79.4%) and PACAP27 (68.4%) with [Ca2+]i rises that could be followed by a refractory plateau (type 1 response), or by a decrease in [Ca2+]i during which somatotropes were responsive to a subsequent PACAP pulse (type II response). Although Ca2+ profiles in response to both peptides were similar, PACAP38-induced [Ca2+]i rises were higher. Somatotrope response to PACAP38 or PACAP27 was markedly reduced by removing extracellular Ca2+, blocking Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC), or inhibiting PKA. Conversely, Ca2+ depletion from intracellular stores or PLC inactivation did not affect the response to PACAP27 but considerably reduced maximal [Ca2+]i induced by PACAP38. We conclude that both peptides stimulate extracellular Ca2+ influx through L-type VSCC by a PKA-dependent mechanism. However, PACAP38 also triggers a PLC-mediated Ca2+ mobilization from intracellular stores, thereby indicating that the two molecular forms of PACAP activate common and distinct second messenger pathways within porcine somatotropes.     

Biocompatibility of a peritoneal dialysis solution with amino acids: histological evaluation in the rabbit

OBJECTIVE: To determine the biocompatibility of a peritoneal dialysis (PD) solution containing amino acids compared to PD solutions containing glucose. DESIGN: The biocompatibility of three dialysis solutions containing 1.1% amino acids, 1.36% glucose, and 3.86% glucose, respectively, was evaluated in vivo in rabbits. METHODS: After 60 days of PD, peritoneal histological changes in rabbits were investigated by light and transmission electron microscopy. The parameters investigated were: (1) mesothelial damage; (2) submesothelial edema; (3) submesothelial cell infiltration; (4) submesothelial fibrosis; and (5) vascular alterations. Semiquantitative evaluations were performed for all the above alterations; quantitative morphometric evaluation was performed for mesothelial damage (cubic transformation of the mesothelium, areas devoid of mesothelium, submesothelial edema) and thickness of peritoneal arteriole walls. RESULTS: (1) Mesothelial damage was practically nonexistent in rabbits dialyzed with the solution containing amino acids, and intermediate and severe with low-glucose and high-glucose solutions, respectively. Both controls and rabbits dialyzed with amino acid solution showed flat continuous mesothelium; rabbits dialyzed with low-glucose solution showed cubic continuous mesothelium; and rabbits dialyzed with high-glucose solution showed cubic discontinuous mesothelium. Cytopathic mesothelial effects were slight with the solution containing amino acids and severe with both the low- and high-glucose solutions. Duplication and thickening of mesothelial basement membrane were never observed. (2) Submesothelial edema showed a worsening trend from controls to rabbits dialyzed with solution containing amino acids, low glucose, and high glucose. (3) No difference in submesothelial infiltration was found between groups. (4) Submesothelial fibrosis was never observed. (5) Vascular alterations were never observed. CONCLUSION: These results are evidence that PD solution with amino acids is more biocompatible than high- and also low-glucose solutions.     

Jugular venous and arterial concentrations of serum S-100B protein in patients with severe head injury: a pilot study

The objective of this study was to analyse the temporal course of the jugular venous-arterial gradient of S-100B protein after severe head injury and the correlation between the absolute concentrations of serum S-100B protein and outcome, CT findings, and clinical variables. Fifteen patients were included in this pilot study. All patients were treated according to a standard therapy protocol targeted to maintain cerebral perfusion pressure. The serum concentration of S-100 protein was measured daily for five consecutive days after injury by a monoclonal two site immunoluminometric assay. Nine patients showed favourable and six unfavourable outcome after 6 months with a mortality rate of 33% (five patients). The mean gradient between jugular venous and arterial blood was 8.2% (p<0.05). Patients showing an unfavourable outcome had significantly higher jugular venous or arterial S-100 values compared with those with a favourable outcome (jugular venous S-100B 2.78 microg/l v 1.22 microg/l, p<0.05; arterial S-100B 2.48 microg/l v 1.19 microg/l, p<0.05). All patients with an initial or secondary increase in S-100B value of >2 microg/l were found to have an unfavourable outcome. S-100B was found to be an independent predictor of outcome after severe head injury. The persisting increase of S-100B for three to five days even in patients with favourable outcome and no signs of secondary insults might reflect continuing damage to the blood-brain barrier or ongoing glial cell death.     

Impact of polyunsaturated fatty acids on human colonic bacterial metabolism: an in vitro and in vivo study

Dietary polyunsaturated fatty acids (PUFA) reduce colonic proliferation and exert a mild laxative effect. We have studied the effect of the highly unsaturated eicosapentaenoic acid ethyl ester (EPA-EE) on the growth and metabolism of colonic bacteria in vitro, and in vivo. For the in vitro study, growth was assessed by viable counts. Bacteroides thetaiotaomicron was significantly inhibited in anaerobic media containing EPA-EE at concentrations > 7 milligrams. Escherichia coli was apparently resistant even at 100 milligrams. For the in vivo study, ten healthy volunteers ingested 18 g EPA-EE/d for 7 d. Stool frequency, 24 h stool weight and whole-gut transit time were assessed together with breath H2 and 14CO2 excretion following oral ingestion of 15 g lactitol labelled with 0.18 MBq [14C]lactitol. The area under the breath-H2-time curve was significantly reduced by EPA-EE, from a control value of 690.3 (SE 94.2) ppm.h to 449.5 (SE 91.7) ppm.h. Percentage dose of 14CO2 excreted, total stool weight and whole-gut transit time were unaltered, being respectively 24 (SE 2)%, 281 (SE 66) g and 45 (SE 4) h with EPA-EE v. control values of 27 (SE 1)%, 300 (SE 89) g and 42 (SE 5) h. It is concluded that dietary supplementation with EPA-EE reduces breath H2 excretion without apparently impairing overall colonic carbohydrate fermentation. The observed reduction may reflect utilization of H2 to hydrogenate the five double bonds of EPA-EE.     

Effect of glutamic acid on broilers given submarginal crude protein with adequate essential amino acids using feeds high and low in potassium

A fully conjugated blood vessel network model (FCBVNM) for calculating tissue temperatures has been developed, tested, and studied. This type of model represents a more fundamental approach to modeling temperatures in tissues than do the generally used approximate equations such as the Pennes'BHTE or effective thermal conductivity equations. As such, this type of model can be used to study many important questions at a more basic level. For example, in the particular hyperthermia application studied herein, a simple vessel network model predicts that the role of counter current veins is minimal and that their presence does not significantly affect the tissue temperature profiles: the arteries, however, removed a significant fraction of the power deposited in the tissue. These more fundamental models can also be used to check the validity of approximate equations. For example, using the present simple model, when the temperatures calculated by the FCBVNM are used for comparing predictions from two approximation equations (a simple effective thermal conductivity and a simple Pennes' bio-heat transfer equation formulation of the same problem) it is found that the Pennes' equation better approximates the FCBVNM temperatures than does the k(eff) model. These results also show that the "perfusion" value (W) in the Pennes' BHTE is not necessarily equal to the "true" tissue perfusion (P) as calculated from mass flow rate considerations, but can be greater than, equal to, or less than that value depending on (1) how many vessel levels are modeled by the BHTE, and (2) the "true" tissue perfusion magnitude. This study uses a simple, generic vessel network model to demonstrate the potential usefulness of such fully conjugated vessel network models, and the associated need for developing and applying more complicated and realistic vascular network models. As more realistic vascular models (vessel sizes, orientations, and flow rates) are developed, the predictions of the fully conjugated models should more closely model and approach the true tissue temperature distributions, thus making these fully conjugated models more accurate and valuable tools for studying tissue heat transfer processes.     

The solution conformations of amino acids from molecular dynamics simulations of Gly-X-Gly peptides: comparison with NMR parameters

The conformations that amino acids can adopt in the random coil state are of fundamental interest in the context of protein folding research and studies of protein-peptide interactions. To date, no detailed quantitative data from experimental studies have been reported; only nuclear magnetic resonance parameters such as chemical shifts and J coupling constants have been reported. These experimental nuclear magnetic resonance data represent averages over multiple conformations, and hence they do not provide unique structural information. I have performed relatively long (2.5 ns) molecular dynamics simulations of Gly-X-Gly tripeptides, surrounded by explicit water molecules, where X represents eight different amino acids with long side chains. From the trajectories one can calculate time averaged backbone chemical shifts and 3J(NH alpha) coupling constants and compare these with experimental data. These calculated quantities are quite close to the experimental values for most amino acids, suggesting that these simulations are a good model for the random coil state of the tripeptides. On the basis of my simulations I predict 3J(alphabeta) coupling constants and present dihedral distributions for the phi, psi, as well as chi1 and chi2 angles. Finally, I present correlation plots for these dihedral angles.     

Toxicity and metabolism of naphthalene: a study with marine larval invertebrates

In 112 plateletphereses done by the Haemonetics blood processor, the comparative effectiveness of ACD formula A, ACD formula B, and 2 per cent citrate in saline was evaluated. With respect to yields of platelets and white blood cells (e.g., lymphocytes), ACD-B was significantly better than the other two. Both ACD-B and 2 per cent citrate gave a much lower incidence of citrate reactions in the donors than were encountered with ACD-A.     

Selective alterations of extracellular brain amino acids in relation to function in experimental portal-systemic encephalopathy: results of an in vivo microdialysis study

Portal-systemic encephalopathy (PSE) is characterized by neuropsychiatric symptoms progressing through stupor and coma. Previous studies in human autopsy tissue and in experimental animal models of PSE suggest that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. To assess this possibility, levels of amino acids were measured using in vivo cerebral microdialysis in frontal cortex of portacaval-shunted rats administered ammonium acetate (3.85 mmol/kg, i.p.) to precipitate severe PSE. Sham-operated rats served as controls. Portacaval shunting resulted in significant increases of levels of extracellular glutamine (threefold, p < 0.001), alanine (38%, p < 0.01), aspartate (44%, p < 0.05), phenylalanine (170%, p < 0.001), tyrosine (140%, p < 0.001), tryptophan (63%, p < 0.001), leucine (75%, p < 0.001), and serine (60%, p < 0.001). Administration of ammonium acetate to sham-operated animals led to a significant increase in extracellular glutamine and taurine content, but this response was absent in shunted rats. The lack of taurine release into extracellular fluid following ammonium acetate administration in portacaval-shunted rats could relate to the phenomenon of brain edema in these animals. Ammonium acetate administration resulted in significant increases in the extracellular concentrations of phenylalanine and tyrosine in both sham-operated and portacaval-shunted rats. Severe PSE was not accompanied by significant increases in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, or serine, suggesting that increased spontaneous release of these amino acids in cerebral cortex is not implicated in the pathogenesis of hepatic coma.     

The sparing action of inorganic sulphate on sulphur amino acids in practical broiler diets: preliminary trials with young chicks

1. In three trials marked responses to Na2SO4 were obtained, both in terms of growth rate and of food/gain ratio. In a fourth trial the chicks responded to graded additions of synthetic methionine but not to inorganic sulphate. 2. The response of the chicks to Na2SO4 depended on the amount of sulphur amino acids in the diets, being absent or difficult to demonstrate in severely deficient diets or in those adequately supplemented, respectively. 3. Sodium sulphate cannot replace more than about 0-05% supplementary methionine. 4. Usually the efficacy of Na2SO4 in improving performance appeared to range from 40% to 60% that of synthetic methionine.