Masaaki Horiguchi

Biosynthesis of 2-aminoethylphosphonic acid in cell-free preparations from tetrahymena

Abstract 1. 1. Incorporation of radioactivity from [32P]orthophosphate and from[3−14C]-phosphoenolpyruvate into 2-aminoethylphosphonic acid (AEPA) was demonstrated in cell-free preparations from Tetrahymena pyriformis GL. 2. 2. Free AEPA became labeled more rapidly than either lipid-bound AEPA or residual AEPA, and the label from [3−14C]phosphoenolpyruvate was preferentially incorporated into the phosphono carbon of AEPA. 3. 3. The rate of incorporation if 32P from orthophosphate and of 14C from phosphoenolpyruvate into AEPA was decreased to a much greater extent by phosphonoacetaldehyde than by 2-amino-3-phosphonopropionic acid. 4. 4. Thus it appears that the major biosynthetic pathway to AEPA is a route through amination of phosphonoacetaldehyde.

Effects of a beta‐adrenergic agonist clenbuterol) on performance, carcase composition, hepatic microsomal mixed function oxidase and antibody production in female broilers treated with or without corticosterone

1. Beta-adrenergic agonist (Clenbuterol, 0.33 mg/kg) and corticosterone (10 mg/kg) were incorporated into a diet based on maize and soybean meal. Their effects on performance, carcase composition, hepatic microsomal mixed function oxidase and antibody production were investigated in female broilers. 2. Dietary corticosterone reduced the titre to sheep red blood cells, while it was unchanged by clenbuterol. 3. Clenbuterol exerted a promoting effect on gain-to-food ratio, carcase protein and hepatic microsomal cytochrome P-450 content. 4. Addition of clenbuterol to the corticosterone-containing diet prevented the increase in abdominal fat weight and uric acid excretion induced by corticosterone, but did not affect total fat mass. 5. The results showed that clenbuterol reduced abdominal rather than carcase fat and prevented protein degradation in the body when chicks were treated with corticosterone. Clenbuterol also influenced the content of cytochrome P-450, but not the humoral immunity.

Metabolism of 2-amino-3-phosphono[3-14C]propionic acid in cell-free preparations of Tetrahymena.

Incubation of 2-amino-3-phosphono[3-14C]propionic acid with cell-free preparations of Tetrahymena pyriformis resulted in incorporation of the radioactivity into phosphoenolpyruvate. No radioactivity was incorporated into phosphoenolpyruvate and 2-phosphonoacetate. Thus, the catabolic pathway of 2-amino-3-phosphonopropionic acid in Tetrahymena appears to be the same route as that found in rats through deamination to produce 3-phosphonopyruvate which is converted to 2-phosphonoacetaldehyde by decarboxylation, followed by both dephosphonylation and amination of the aldehyde to give acetaldehyde and 2-aminoethylphosphonic acid, respectively.

Species differences between chickens and rats in chemical properties of adipose tissue lipoprotein lipase

Chemical characterization of chicken and rat lipoprotein lipase (LPL) was carried out following purification of LPL. Molecular weight and isoelectric point of both purified enzymes were determined to be 60 KDa and pH 4, while optimum temperature and pH to yield the maximal activity were about 37 degrees C and pH 8.5. Metallic ions, NaCl and protamine sulfate reduced, and heparin increased, both LPL activities. Michaelis constants for LPLs determined with triolein emulsion as the substrate were 0.98 and 1.57, and those of Vmax were 379.2 and 181.3, in chickens and rats, respectively. Triton WR-1339 caused mixed-type inhibition in rat, but inhibited chicken LPL noncompetitively. In LPLs of chickens and rats, values of Ki were 66.7 and 36.4 with triolein emulsion as the substrate, and 832.4 and 66.0 with respective VLDL as the substrate. These results show species difference between chickens and rats in the affinity to lipoproteins of LPL and inhibition of LPL by Triton WR-1339.

SPECIES DIFFERENCES BETWEEN CHICKS AND RATS IN INHIBITION OF LIPOPROTEIN HYDROLYSIS BY TRITON WR-1339

To identify the species differences in lipoprotein hydrolysis, plasma lipoproteins and lipoprotein lipase (LPLase) prepared from both rats and chicks were incubated in vitro in the presence of Triton WR-1339 at concentrations up to 500 micrograms/ml. Rate of lipoprotein hydrolysis by LPLase declined gradually with an increase of Triton concentration irrespective of differences in sources in the lipoprotein and LPLase. At 250 micrograms/ml Triton, the lipoprotein hydrolysis was inhibited by 90% in rats but was only inhibited by < 20% in chicks. Lipoprotein hydrolysis inhibited by Triton was partly recovered by an increase of LPLase concentration, and extents of the recovery were prominent either when rat lipoprotein was provided in place of chick lipoprotein and when rat LPLase rather than chick LPLase was used as the enzyme source for each lipoprotein. These results suggest species differences between chicks and rats with regard to the affinity of Triton to LPLase and lipoprotein and/or the chemical characteristics of both LPLase and lipoprotein.

Biosynthesis of 3-phosphonopyruvic acid in cell-free preparations of Tetrahymena pyriformis GL

Abstract The existence of 3-phosphonopyruvic acid in cell-free preparations of Tetrahymena pyriformis GL incubated with phospho enol pyruvate has been confirmed by its conversion to a 2,4-dinitrophenylhydrazone and analysis on TLC and HPLC. The effects of some compounds on the biosynthesis of phosphonopyruvic acid have been studied.

L-tryptophan alleviates fatty liver and modifies hepatic microsomal mixed function oxidase in laying hens.

1. Three experiments were conducted to study the effect of dietary L-tryptophan supplementation (250-1000 ppm) on lipid accumulation, an occurrence of hemorrhages and microsomal mixed function oxidase in the liver of laying hens. 2. Dietary L-tryptophan supplementation resulted in significant decreases in hepatic lipids, in particular triglyceride, and occurrence of hemorrhage in laying hens. 3. Hepatic lipid accumulation by estrogen injection in starved-refed growing chicks decreased as dietary tryptophan content increased. 4. Supplementation of L-tryptophan at 1000 mg/kg diet enhanced alanine aminotransferase activity in the hepatic tissue and at 500 mg/kg diet, increased cytochrome b5, a component of the mixed function oxidase, in the hepatic microsomes. 5. These results demonstrate that L-tryptophan alleviates fatty liver in laying hens and modifies microsomal mixed function oxidase in the liver.

Metabolizable energy value and effect on amino acid availability of medium-chain triglycerides in diets fed to chickens of different ages

Abstract Three experiments were conducted to determine the energy value and digestibility of medium-chain triglycerides (MCT) in chickens. Triacylglycerol with capric acids (C10-TG) and triacylglycerol with capric and caprylic acids (C10C8-TG) were provided as the MCT sources. Sixty 3-day-old broiler chicks and 25 adult roosters were provided for the determination of nitrogen-corrected true metabolizable energy (TMEn) of MCT. Forty 3-week-old broiler chicks were provided for the determination of nitrogen-corrected apparent metabolizable energy (AMEn) of MCT. True amino acid availability of amino acids (TAAA) of a diet with MCT was determined using adult roosters. The TMEn of C10-TG (37.11–38.45 MJ kg −1 ) was not significantly different from that of yellow grease (YG, 39.29–40.54 MJ kg −1 ), a long-chain triglycerides (LCT), in 3-day-old chicks. In 3-week-old chickens, the AMEn of C10-TG (32.04 MJ kg −1 ) was comparable with that of YG (32.00 MJ kg −1 ). The TMEn (36.02–36.44 MJ kg −1 ) and AMEn (33.76 MJ kg −1 ) of C10C8-TG were higher to a certain extent than YG (AMEn: 32.00 MJ kg −1 ) and C10-TG (TMEn: 25.90–31.12 MJ kg −1 ; AMEn: 32.04 MJ kg −1 ). Digestibility of lipids in the basal diet (68.1%) and that of C10-TG (96.3–98.5%) was low in 3-day-old chicks as compared with those in roosters (78.8% and 100.0–107.9%). The digestibility of MCT (96.3–107.9%) was higher than YG (94.0–94.6%), irrespective of the age of chickens. TAAA was not influenced by the inclusion of MCT in the diet. It is suggested that the digestibility of C10-TG and C10C8-TG is higher than that of YG and the TMEn and AMEn values are comparable with that of YG. Therefore, MCT is a potential fat source for chickens, particularly for very young chicks.

Metabolism of 2-amino-3-phosphonopropionic acid in rats

The metabolism of 2-amino-3-phosphono-[2-(14)C]propionic acid or 2-amino-3-phosphono-[3-(14)C]propionic acid in rats was studied in vivo and in vitro. The radioactivity in expired CO2 from the [3-(14)C]-labelled compound indicated the cleavage of the carbon-phosphorus (C-P) bond. A small amount of the [2-(14)C]-labelled compound and the [3-(14C]-labelled compound was incorporated into 2-aminoethylphosphonic acid, and polar lipid of the liver and kidney contained the 2-aminoethylphosphonic acid. The 2-amino-3-phosphonopropionic acid was not detected at the lipid level. Incorporation of the [3-(14)C]-labelled compound into a variety of metabolites including 3-phosphonopyruvic acid and 2-phosphonoacetaldehyde suggests the transamination reaction as a decomposition mechanism of 2-amino-3-phosphonopropionic acid in mammals.

Sex-related differences of hepatic microsomal mixed function oxidase system and antibody production in broilers implanted with corticosterone and/or fed ascorbic acid.

1. Most of the components of the mixed function oxidase (MFO) in hepatic microsomes were reduced by corticosterone implants, and the degree of the reduction in females and at an older age was greater than those in males and at a younger age. 2. Ascorbic acid (AA) prevented the reduction in the MFO caused by corticosterone implants. 3. The activities of aniline hydroxylase and aminopyrine N-demethylase were enhanced by corticosterone implants regardless of AA supplementation. 4. The activity of NADPH-cytochrome c reductase in male broiler was greater than that in females under normal conditions. 5. Corticosterone implants and dietary AA had less influence on the antibody production, especially to T-cell dependent antigen.