Evaluation of the pepsin digestibility assay for predicting amino acid digestibility of meat and bone meals.

Sixteen meat and bone meal (MBM) samples were obtained and selected from various company plants to provide a wide range in pepsin nitrogen digestibility values. Pepsin digestibility was determined using either 0.02 or 0.002% pepsin. Amino acid (AA) digestibility of the 16 MBM samples was then determined using a precision-fed cecectomized rooster assay. The 0.02% pepsin digestibility values were numerically higher than the 0.002% pepsin values. The values varied from 77 to 93% for 0.02% pepsin and from 67 to 91% for 0.002% pepsin. The rooster AA digestibility results showed a wide range of values among MBM samples mostly due to the 4 samples having lowest and highest AA digestibility. A precision-fed broiler chick ileal AA digestibility assay confirmed that there were large differences in AA digestibility among the MBM samples having the lowest and highest rooster digestibility values. Correlation analyses between pepsin and AA digestibility values showed that the correlation values (r) were highly significant (P < 0.0001) for all AA when all 16 MBM samples were included in the analysis. However, when the MBM samples with the 2 lowest and the 2 highest rooster digestibility values were not included in the correlation analyses, the correlation coefficient values (r) were generally very low and not significant (P > 0.05). The results indicated that the pepsin nitrogen digestibility assay is only useful for detecting large differences in AA digestibility among MBM. There also was no advantage for using 0.02 versus 0.002% pepsin.

Discovery of OT4003, a novel, potent, and orally active cys-LT1 receptor antagonist.

The present paper describes the structural modifications leading to the discovery of a new series of quinoline-containing cys-LT1 receptor (LTD4 receptor) antagonists. A structural optimization with respect to the in vitro receptor binding, the in vivo brochoconstriction, and the toxicological effect in the form of peroxisomal proliferation was performed in order to achieve the target compound OT4003. OT4003 ((S)-(+)-E-2-(3-(2-(7- chloroquinolin-2-yl)ethenyl)phenylaminomethyl)-phenoxyl++ +-hexanoic acid) was found to be a potent and selective inhibitor of [3H]LTD4 specific binding to guinea pig lung membranes (IC50 2.4 +/- 1.0 nM), and also a potent, orally active, antagonist of LTD4 induced bronchoconstriction in guinea pigs [ED50 0.14 (ED16 0.1-ED84 0.4) mg/kg; 4 h pretreatment]. The enantiomerically pure OT4003 was prepared using a short convergent synthesis, including an enzymatic resolution step.

Novel antibody coating of a magnetizable solid phase for use in enzyme immunoassays.

Novel kinds of magnetizable particles have been prepared using the interaction between the complexing groups of phosphonic acid and polyvalent metal ions on the surface of Fe3O4 particles. After modification of monoclonal and polyclonal antibodies by the bifunctional chelating agent 2-(4-diazophenyl)-1-hydroxyethane-1,1-bisphosphonic acid, antibodies were immobilized at a ratio of 5-10 mg antibody molecules per ml Fe3O4 particles without loss of immunological reactivity. Very sensitive and fast immunoenzymometric assays for the quantitative determination of human interferon-alpha 1 and mouse immunoglobulins were developed using such particles. The advantages of the method include immobilization of proteins on magnetizable carriers without chemical modification of the carrier and the short assay time compared to conventional immunoenzymometric assays.

Recombinant human interleukin-8 is a potent activator of canine neutrophil aggregation, migration, and leukotriene B4 biosynthesis.

Interleukin-8 (IL-8), formerly known as NAP-1, is formed by a variety of cells upon stimulation with IL-1 or tumor necrosis factor (TNF). The biologic activity of the cytokine involves activation of almost every neutrophil function studied so far in different species. In the present study, we compared the effects of recombinant human IL-8 (rIL-8) and the lipid mediators, leukotriene B4 (LTB4) and platelet-activating factor (PAF), on neutrophil functions in dogs. All three chemotactic factors induced neutrophil aggregation and chemotaxis, with rIL-8 being far more potent than LTB4 and PAF. The migration induced by rIL-8 was significantly greater than that observed towards LTB4 and PAF. In the aggregation assay, rIL-8 was shown for the first time to be a potent stimulant. The aggregation response was more persistent than that obtained with LTB4 and PAF and the potency of rIL-8 was greater. An intradermal dose-response study showed that rIL-8 is an extremely potent inducer of selective neutrophil infiltration in canine skin. The infiltration was more pronounced than following injection of LTB4 or PAF. It was proposed that the superior effect of rIL-8 was caused by a synergistic effect between injected rIL-8 and LTB4, which was shown to be produced in biologically active amounts by canine neutrophils stimulated with rIL-8. From a therapeutic point of view, the simultaneous presence of rIL-8 and LTB4 in inflammatory skin diseases highlights the need to develop drugs that inhibit the production and/or effect of both mediators.

Inhibition of leukotriene biosynthesis and polymorphonuclear leukocyte functions by orally active quinolylmethoxyphenylamines.

The N-substituted quinolylmethoxyphenylamines, ETH603, ETH615 and ETH647, inhibited the formation of LTB4 in rat peritoneal leukocytes, human peripheral polymorphonuclear leukocytes and canine whole blood. In rat and human cells, the compounds also inhibited the formation of 5-HETE and stimulated the synthesis of 15-HETE. In rat leukocytes, the compounds were 15-30 times more potent inhibitors of LTB4 synthesis than nordihydroguaiaretic acid, but in canine whole blood they were significantly less potent, possibly due to protein binding. However, after oral administration of the compounds to dogs a long-lasting inhibition of LTB4 production in peripheral blood was observed at serum concentrations much lower than those required in vitro. Furthermore, the compounds inhibited the LTB4-directed chemotaxis and the phagocytosis of C. albicans blastospores by canine polymorphonuclear leukocytes both in vitro and following oral administration. The calcium ionophore A23187-induced release of LTB4 in the peritoneal cavity of rats was also inhibited by systemic administration of the compounds. We therefore conclude that these novel quinolines are orally active 5-lipoxygenase inhibitors which may accumulate in inflammatory cells in vivo, leading to potent inhibition of leukotriene biosynthesis and cell function.

A novel leukotriene D4/E4 antagonist, SR2640 (2-[3-(2-quinolylmethoxy)phenylamino]benzoic acid).

We have studied the leukotriene antagonistic activity of a novel compound, SR2640 (2-[3-(2-quinolylmethoxy)phenylamino]benzoic acid), in vitro and in vivo. SR2640 inhibited LTD4- but not histamine-induced contractions of guinea-pig ileum and trachea in a concentration-dependent manner. Schild plot analysis of tracheal LTD4 antagonism yielded a pA2 value of 8.7 and a slope not different from unity. SR2640 concentration dependently inhibited the binding of 0.4 nM [3H]LTD4 to guinea-pig lung membranes with an IC50 value of 23 nM. The curve was parallel to that of unlabelled LTD4 (IC50 = 2.2 nM). SR2640 was equally effective in antagonizing LTD4 and LTE4, but was much less potent in reducing LTC4-induced ileum contractions. In vivo, SR2640 in the dose range 0.03-1.00 mg/kg shifted the dose-response curve for guinea-pig bronchoconstriction induced by intravenous LTD4 administration to the right at a rate proportional to the dose of SR2640, without reducing the maximum attainable obstruction: the slope of the Schild plot was 0.99. SR2640 (1 mg/kg) also caused a significant inhibition of antigen-induced bronchoconstriction in anaesthetized guinea-pigs pretreated with pyrilamine, indomethacin, propranolol and suxamethonium. In conclusion, SR2640 appears to be a potent and selective competitive LTD4/LTE4 antagonist, and may be useful in elucidating the role of leukotrienes in human asthma.

Atrial content and plasma levels of atrial natriuretic peptides in rats with chronic renal failure.

The possible role of atrial natriuretic peptides (ANP) for the adaptive changes in renal Na excretion during chronic renal failure was studied in 5/6 nephrectomized (NX) rats maintained on a normal (100 mmol/kg) and a high (800 mmol/kg) Na diet. Atrial content of natriuretic substances was determined by bioassay and plasma ANP by radioimmunoassay. Nephrectomized rats showed a twofold increase in plasma ANP irrespective of their Na intake. Atrial ANP content was increased by high Na diet but unchanged by NX. Nephrectomized rats maintained on high Na diet showed partial depletion of atrial ANP stores. There were no significant changes in the volume fraction of atrial granules determined. The results suggest that ANP is involved in the regulation of renal Na excretion during chronic renal failure and acute Na loading; other mechanisms are probably involved in the adaption to chronic Na loading.

Enzyme electrodes and their application.

Starting from the state of the art, principles for improving the analytical characteristics of enzyme electrodes are discussed. Coupling of appropriate amperometric electrode processes with enzyme systems, e.g. urease or aminopeptidases, results in a simplification of operation. Optimal sample frequencies are realized on the basis of enzyme membranes, with both a small characteristic diffusion time and a high enzyme activity, applied in a well-designed sample-processing system. Coupled enzyme reactions of the sequence or competition type are successfully used for extension to new analytes, e.g. inhibitors, cofactors or alternative substrates. Cyclization of the analyte enhances the sensitivity of enzyme electrodes to the nanomolar concentration range. Enzymic anti-interference layers are a tool for improving the sensor specificity. The operational characteristics of enzyme electrodes are thus adaptable to any given analytical problem.

Enzymatic determination of isocitrate by amperometric monitoring of the rate of oxygen consumption.

A coupled enzymatic method elaborated for NAD+-dependent dehydrogenases has been adapted for NADP+-dependent isocitrate dehydrogenase, in combination with amperometric measurements. The isocitrate dehydrogenase activity dependent linearly on the isocitrate concentration in the range 0-2 X 10(-4) M. Application of this method affords a sensitive estimation of isocitrate even in turbid liquids such as fermentation broths.

Changes in plasma lipoproteins from first day to third week of life in healthy breast-fed infants. I. Lipid and protein composition of lipoproteins.

Plasma lipoproteins were measured in seven healthy breast-fed infants three times during the first and on the third, sixth and twenty-first days of life. Plasma total beta-lipoprotein, very low density and low density lipoprotein increased three times during the first week of life. Plasma total apolipoprotein A-I and high density lipoprotein increased twice between the first and third week of life. These dramatic changes in plasma lipoprotein concentrations during the neonatal period can best be explained by the effect of feeding.

Changes in plasma lipoproteins from first day to third week of life in healthy breast-fed infants. II. Fatty acid composition of lipoprotein triglyceride- and cholesterol esters.

The fatty acid composition of plasma lipoprotein-triglyceride (TG) and -cholesterol ester (CE) was studied in seven healthy breast-fed infants three times during the first day and on the third, sixth and twenty-first days of life. The major finding was a two-fold increase in the linoleic acid weight percent of plasma lipoprotein-TG and -CE between the first and third weeks of life. This increase could best be explained by the ingestion of breast-milk rich in linoleic acid.

Enzyme electrode for urea with amperometric indication: Part I--Basic principle.

A novel method for amperometric determination of substrates of hydrolytic enzymes has been developed. As an example the pH dependence of electrochemical oxidation of hydrazine in the Tafel region was combined with the urease catalysed splitting of urea to construct an amperometric membrane electrode for urea. The characteristic features of this sensor are: a linear dependence of the current of hydrazine oxidation on hydrogen ion concentration (as opposed to the logarithmic response of potentiometric sensors), linear urea concentration-signal relationship between 0.8 and 35 mmol/litre sample concentration, a response time of about 20 s, a relative standard deviation of 1% and measuring frequency up to 40 samples/h with an electrode operational stability of 2 weeks. Calibration curves for aqueous urea solutions are given as functions of starting pH, urease and hydrazine concentration and the potential dependence of the signal was determined.

Acyl group distributions in tissue lipids of rats fed evening primrose oil (gamma -linolenic plus linoleic acid) or soybean oil (alpha-linolenic plus linoleic acid).

Three groups of rats were fed diets with either 10 weight percent (wt%) of evening primrose oil, safflower oil or soybean oil for 11 weeks. Diets contained 7.1 wt% linoleic acid + 0.8 wt% gamma-linolenic acid, 7.6 wt% linoleic acid, or 5.3 wt% linoleic acid + 0.7 wt% alpha-linolenic acid, respectively. In liver mitochondria as well as in heart, dietary gamma-linolenic acid did not affect the fatty acid profiles of phosphatidylcholines (PC), phosphatidylethanolamines (PE) or cardiolipins (CL), whereas dietary alpha-linolenic acid caused an increased formation of (n-3) polyunsaturated fatty acids (PUFA). The liver delta 6- and delta 5-desaturase activities determined in vitro were not affected by the dietary fats. In brain PE, which are rich in C22- and C20-(n-3) PUFA, as well as in testes PC and PE, which are rich in (n-6) PUFA, no effects were found from a partial replacement of dietary linoleic acid with gamma-linolenic acid or alpha-linolenic acid. In kidney PC, PE, phosphatidylinositol (PI) and CL, 20:3(n-6) was moderately elevated to ca. 1% following intake of gamma-linolenic acid, whereas partial replacement of linoleic acid with alpha-linolenic acid was followed by increased deposition of 22:6(n-3) in PC and PE of testes and kidney. Thus, no general effect of evening primrose oil on the content of (n-6) PUFA in rat tissue phospholipids was observed, whereas a significant incorporation of gamma-linolenic acid into liver and adipose tissue triglycerides was found.

Effect of dietary fats on the delta 6- and delta 5-desaturation of fatty acids in rat liver microsomes.

Rats were given diets containing (% dietary energy): 46 arachis oil (AO), 36 partially-hydrogenated arachis oil (HAO) + 10 AO, 36 partially-hydrogenated marine oil (HMO) + 10 AO, or 46 of a combination of rape-seed oils high and low in erucic acid (RSO + LERSO). In the liver microsomes the content of arachidonic acid (20:4 omega 6) was reduced in the groups given HAO + AO and HMO + AO. The rates of delta 6-desaturation of linoleic acid into gamma-linolenic acid (18:3 omega 6) and of delta 5-desaturation of dihomo-gamma-linolenic acid into arachidonic acid were studied in vitro at two substrate levels: a high substrate level reflecting maximal microsomal desaturase activity in rat liver and a low substrate level reflecting desaturase activity under physiological conditions. Dietary HAO, rich in 18:1 isomers, suppressed the delta 6-desaturase activity but not the delta 5-desaturase activity. Dietary HMO, rich in 18:1, 20:1 and 22:1 isomers, reduced both delta 6- and delta 5-desaturase activities.

Acyl group distributions in tissue lipids of rats fed evening primrose oil (λ-linolenic plus linoleic acid) or soybean oil (α-linolenic plus linoleic acid).

Three groups of rats were fed diets with either 10 weight percent (wt%) of evening primrose oil, safflower oil or soybean oil for 11 weeks. Diets contained 7.1 wt% linoleic acid +0.8 wt% γ-linolenic acid, 7.6 wt% linoleic acid, or 5.3 wt% linoleic acid +0.7 wt% α-linolenic acid, respectively. In liver mitochondria as well as in heart, dietary γ-linolenic acid did not affect the fatty acid profiles of phosphatidylcholnes (PC), phosphatidylethanolamines (PE) or cardiolipins (CL), whereas dietary α-linolenic acid caused an increased formation of (n-3) polyunsaturated fatty acids (PUFA). The liver Δ6- and Δ5-desaturase activities determined in vitro were not affected by the dietary fats. In brain PE, which are rich in C22- and C20-(n-3) PUFA, as well as in testes PC and PE, which are rich in (n-6) PUFA, no effects were found from a partial replacement of dietary linoleic acid with γ-linolenic acid or α-linolenic acid. In kidney PC, PE, phosphatidylinositol (PI) and CL, 20∶3(n-6) was moderately elevated to ca. 1% following intake of γ-linolenic acid, whereas partial replacement of linoleic acid with α-linolenic acid was followed by increased deposition of 22∶6(n-3) in PC and PE of testes and kidney. Thus, no general effect of evening primrose oil on the content of (n-6) PUFA in rat tissue phospholipids was observed, wheras a significant incorporation of γ-linolenic acid into liver and adipose tissue triglycerides was found.

Influence of partially hydrogenated vegetable and marine oils on lipid metabolism in rat liver and heart.

Partially hydrogenated marine oils containing 18:1-, 20:1- and 22:1-isomers and partially hydrogenated peanut oil containing 18:1-isomers were fed as 24-28 wt% of the diet with or without supplement of linoleic acid. Reference groups were fed peanut, soybean, or rapeseed oils with low or high erucic acid content. Dietary monoene isomers reduced the conversion of linoleic acid into arachidonic acid and the deposition of the latter in liver and heart phosphatidylcholine. This effect was more pronounced for the partially hydrogenated marine oils than for the partially hydrogenated peanut oil. The content of trans fatty acids in liver phospholipids was similar in groups fed partially hydrogenated fats. The distribution of various phospholipids in heart and liver was unaffected by the dietary fat. The decrease in deposition of arachidonic acid in rats fed partially hydrogenated marine oils was shown in vitro to be a consequence of lower delta 6-desaturase activity rather than an increase in the peroxisomal beta-oxidation of arachidonic acid. The lower amounts of arachidonic acid deposited may be a result of competition in the delta 6-desaturation not only from the C22- and C20-monoenoic fatty acids originally present in the partially hydrogenated marine oil, but also from C18- and C16-monoenes produced by peroxisomal beta-oxidation of the long-chain fatty acids.

[Preparation and properties of immobilized glucose oxidase]. / Darstellung und Eigenschaften trägerfixierter Glukoseoxydase.

Glucose oxidase from Penicillium notatum was immobilized by covalent, adsorptive or ion exchange attachment to insoluble carriers. The yields of immobilization using Spherons, DEAE-Sephadex, DEAE-cellulose and porous glass carriers are compared. Methods used for the estimation of kinetic parameters Km and kcat are described and results obtained for GOD in solution and in immobilized form are given. Investigations about the dependence of the enzymatic activity on pH, temperature and storage serve for the further characterization of the GOD-derivatives. There are no significant changes in the functional behaviour of the enzyme due to immobilization on selected carriers. The results are discussed with regard to application of carrier bound GOD in enzyme reactors.